IgG bispecific antibodies and processes for preparation

ABSTRACT

The present invention provides fully IgG bi-specific antibodies comprising designed residues in the interface of the heavy chain-heavy chain (C H 3/C H 3) domains, processes for preparing said fully IgG bi-specific antibodies, and nucleic acids, vectors and host cells encoding the same.

CROSS-REFERENCE OF RELATED APPLICATIONS

This application is a national stage entry of the PCT Patent Application PCT/US2016/014313 filed on Jan. 21, 2016, which claims priority to and the benefit under 35 U.S.C. 119(e) to U.S. Provisional Patent Application No. 62/106,494, filed on Jan. 22, 2015.

BACKGROUND OF THE INVENTION

Antibody therapies represent an ever increasing segment of the global pharmaceutical market. Approved antibody-based products include treatments for cancer, autoimmune disorders (e.g. rheumatoid arthritis), infectious diseases, cardiovascular disease and many other disorders. However, to improve patient outcomes, perturbation of multiple therapeutic targets or biochemical pathways is often desired. In this context, antibody therapy has limitations.

Co-administration of two or more antibody therapies requires multiple injections or, alternatively, a single injection of a co-formulation of two different antibody compositions. While multiple injections permit flexibility in dose and timing of administration, the inconvenience and discomfort associated with multiple injections may reduce patient compliance. While a co-formulation of multiple antibody agents would permit fewer injections, the difficulty and/or expense associated with designing a suitable pharmaceutical formulation that provides the necessary stability and bioavailability, for each antibody ingredient, may be prohibitive. Further, any treatment regime which entails administration of separate antibody agents will incur added manufacturing and regulatory costs associated with the development of each individual agent.

Bispecific antibodies (BsAbs)—single agents capable of binding to two distinct antigens or epitopes—have been proposed as a means for addressing the limitations attendant with co-administration or co-formulation of separate antibody agents. BsAbs integrate the binding activities of two separate antibody therapeutics into a single agent, thus providing a potential cost and convenience benefit to the patient. In some circumstances, BsAbs may also elicit synergistic or novel activities beyond what an antibody combination can achieve.

Recombinant DNA technologies have enabled the generation of multiple BsAb formats. For example, single chain Fv (scFv) fragments composed of antigen recognition domains (i.e., heavy chain variable (V_(H)) and light chain variable (V_(L)) domains) tethered by flexible or structured linkers, taken from existing monoclonal antibody (MAb) therapeutics or discovered by in vitro screening methodologies, have been used as building blocks for BsAb generation. In this context, an scFv fragment(s) which binds a particular antigen can be linked to another moiety, for example a separate scFv or an IgG MAb which binds a separate antigen, to form multi-valent BsAb. However, a limitation with the use of scFvs is that they lack the archetypical Fab architecture which provides stabilizing interactions of the heavy chain (HC) and light chain (LC) constant domains (i.e., C_(H)1 and C_(L), respectively) which can improve thermal stability or solubility, or reduce the potential for aggregation.

The ability to generate bispecific antibodies retaining the full IgG antibody architecture has long been a challenge in the field of antibody engineering. One approach for generating fully IgG bispecific antibodies entails co-expression of nucleic acids encoding two distinct HC-LC pairs which, when expressed, assemble to form a single antibody comprising two distinct Fabs. However, to achieve efficiency in manufacturing, each of the expressed polypeptides of the distinct HC-LC pairs must assemble with its cognate polypeptide with good specificity to reduce generation of mis-matched Fab by-products. In addition, the two distinct HCs must heterodimerize in assembly to reduce generation of mono-specific antibody by-products. Fab interface designs which promote assembly of particular HC-LC pairs have recently been described. (See Lewis, et al. (2014), Nat. Biotechnol., 32; 191-198; and Published PCT Applications WO2014/150973 and WO2014/0154254) in addition, procedures for directing assembly of particular HC-HC pairs by introducing modifications into regions of the HC-HC interface have also been disclosed in the art. (See Klein et al., mAbs; 4(6); 1-11 (2012); Carter et al., J. Immunol. Methods; 248; 7-15 (2001); Gunasekaran, et al, J. Biol. Chem.; 285; 19637-19646 (2010); Zhu et al., Protein Sci.; 6: 781-788 (1997); and Igawa et al., Protein Eng. Des. Sel.; 23; 667-677 (2010)). However, there remains a need for alternative methods for generating fully IgG BsAbs.

DETAILED DESCRIPTION

In accordance with the present invention, HC-HC interface designs and processes have been identified for improving assembly of fully IgG bispecific antibodies. The designs and processes of the present invention achieve improved heterodimerization of distinct heavy chains by introducing specific mutations in the C_(H)3 domain of IgG1, IgG2 or IgG4 constant regions, and may be combined with known methods for improving HC-LC specific assembly, thus facilitating assembly of fully IgG BsAbs. In particular, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or IgG4 constant region, wherein said first human IgG1 or IgG4 constant region comprises an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or IgG4 constant region, wherein said second human IgG1 or IgG4 constant region comprises a valine or methionine substituted at residue 366 (or a valine or methionine at residue 366) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

More particularly, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or IgG4 constant region, wherein said first human IgG1 or IgG4 constant region comprises an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (h) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or IgG4 constant region, wherein said second human IgG1 or IgG4 constant region comprises a valine substituted at residue 366 (or a valine at residue 366) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

More particularly, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 or IgG4 constant region comprises an alanine substituted at residue 407 (or an alanine at residue 407) and a methionine substituted at residue 399 (or a methionine at residue 399) of the C_(H)3 domain; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or IgG4 constant region, wherein said second human IgG1 or IgG4 constant region comprises a valine substituted at residue 366 (or a valine at residue 366) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

More particularly, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or IgG4 constant region, wherein said first human IgG1 or IgG4 constant region comprises an alanine substituted at residue 407 (or an alanine at residue 407), a methionine substituted at residue 399 (or a methionine at residue 399) and an aspartic acid substituted at residue 360 (or an aspartic acid at residue 360) of the C_(H)3 domain; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or IgG4 constant region, wherein said second human IgG1 or IgG4 constant region comprises a valine substituted at residue 366 (or a valine at residue 366), a valine substituted at residue 409 (or a valine at residue 409), and an arginine substituted at residues 345 and 347 (or an arginine at residues 345 and 347) of the C_(H)3 domain; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

As another particular embodiment, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or IgG4 constant region, wherein said first human IgG1 or IgG4 constant region comprises an aspartic acid substituted at residue 357 (or an aspartic acid at residue 357), a glutamine substituted at residue 364 (or a glutamine at residue 364) and an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or IgG4 constant region, wherein said second human IgG1 or IgG4 constant region comprises a serine substituted at residue 349 (or a serine at residue 349), a valine substituted at residue 366 (or a valine at residue 366), a tyrosine substituted at residue 370 (or a tyrosine at residue 370) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

Another particular embodiment of the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or IgG4 constant region, wherein said first human IgG1 or IgG4 constant region comprises an aspartic acid substituted at residue 357 (or an aspartic acid at residue 357), a glutamine substituted at residue 364 (or a glutamine at residue 364) and an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or IgG4 constant region, wherein said second human IgG1 or IgG4 constant region comprises a serine substituted at residue 349 (or a serine at residue 349), a methionine substituted at residue 366 (or a methionine at residue 366), a tyrosine substituted at residue 370 (or a tyrosine at residue 370) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

The present invention further provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or IgG4 constant region, wherein said first human IgG1 or IgG4 constant region comprises an aspartic acid substituted at residue 357 (or an aspartic acid at residue 357), an arginine substituted at residue 364 (or an arginine at residue 364) and an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or IgG4 constant region, wherein said second human IgG1 or IgG4 constant region comprises a serine substituted at residue 349 (or a serine at residue 349), a valine substituted at residue 366 (or a valine at residue 366), a tyrosine substituted at residue 370 (or a tyrosine at residue 370) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

Yet another particular embodiment provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or IgG4 constant region, wherein said first human IgG1 or IgG4 constant region comprises a glycine substituted at residue 356 (or a glycine at residue 356), an aspartic acid substituted at residue 357 (or an aspartic acid at residue 357), a glutamine substituted at residue 364 (or a glutamine at residue 364) and an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or IgG4 constant region, wherein said second human IgG1 or IgG4 constant region comprises a serine substituted at residue 349 (or a serine at residue 349), a valine substituted at residue 366 (or a valine at residue 366), a tyrosine substituted at residue 370 (or a tyrosine at residue 370) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

The present invention further provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or IgG4 constant region, wherein said first human IgG1 or IgG4 constant region comprises a glycine substituted at residue 356 (or a glycine at residue 356), an aspartic acid substituted at residue 357 (or an aspartic acid at residue 357), a glutamine substituted at residue 364 (or a glutamine at residue 364) and an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or IgG4 constant region, wherein said second human IgG1 or IgG4 constant region comprises a serine substituted at residue 349 (or a serine at residue 349), a methionine substituted at residue 366 (or a methionine at residue 366), a tyrosine substituted at residue 370 (or a tyrosine at residue 370) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

As another particular embodiment, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or IgG4 constant region, wherein said first human IgG1 or IgG4 constant region comprises an aspartic acid substituted at residue 357 (or an aspartic acid at residue 357), an arginine substituted at residue 364 (or an arginine at residue 364) and an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or IgG4 constant region, wherein said second human IgG1 or IgG4 constant region comprises a serine substituted at residue 349 (or a serine at residue 349), a methionine substituted at residue 366 (or a methionine at residue 366), a tyrosine substituted at residue 370 (or a tyrosine at residue 370) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

As additional particular embodiments of the present invention, IgG BsAbs are provided which comprise first and second heavy chains comprising human IgG1 or human IgG4 constant regions, wherein each of said human IgG1 or human IgG4 constant regions comprise C_(H)2-C_(H)3 segments of a particular amino acid sequence. Thus, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:7; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:42; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

As another particular embodiment, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:38; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:42; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

The present invention also provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:39; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:43; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

As another particular embodiment, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:40; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:44; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)), and a second constant domain (C_(L)).

As another particular embodiment, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:41; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)), and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:44; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)), and a second constant domain (C_(L)).

Yet another particular embodiment provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:40; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:46; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

The present invention also provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ NO:40; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:45; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

Another embodiment provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:41; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:45; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

Further, the present invention also provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:41; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:46; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

Further, the present invention also provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ NO:60; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:65; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

As another particular embodiment, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:61; (b) a first light chain, wherein said first light chain comprises a first variable domain (q) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ NO:65; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

The present invention also provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:62; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:66; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

As another particular embodiment, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:63; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:67; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

As another particular embodiment, the present invention provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:64; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:67; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

Yet another particular embodiment provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:63; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:69; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

The present invention also provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:63; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:68; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

Another embodiment provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:64; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:68; and (d) a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

Further, the present invention also provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ NO:64; (b) a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:69; and (d) a second light chain, wherein said second light chain comprises a second variable domain and a second constant domain (C_(L)).

As an even more particular embodiment, the present invention combines C_(H)3 domain designs in the IgG1, IgG2 or IgG4 constant regions with Fab designs as described in Lewis et al. (2014) and WO2014/150973. In particular, the present invention provides an IgG bispecific antibody according to any one of the afore-mentioned IgG bispecific antibodies, wherein: (a) one of said first or second heavy chains further comprises a variable domain (V_(H)) comprising a lysine substituted at residue 39 (or a lysine at residue 39) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and a human IgG1 or human IgG4 C_(H)1 domain comprising an alanine substituted at residue 172 (or an alanine at residue 172) and a glycine substituted at residue 174 (or a glycine at residue 174); (b) one of said first or second light chains comprises a kappa variable domain (V_(L)), comprising an arginine substituted at residue 1 (or an arginine at residue 1) and an aspartic acid substituted at residue 38 (or an aspartic acid at residue 38), and a constant domain (C_(L)) comprising a tyrosine substituted at residue 135 (or a tyrosine at residue 135) and a tryptophan substituted at residue 176 (or a tryptophan at residue 176); (c) the other of said first or second heavy chains further comprises a variable domain (V_(H)) comprising a tyrosine substituted at residue 39 (or a tyrosine at residue 39) and a WT human IgG1 or human IgG4 C_(H)1 domain; and (d) the other of said first or second light chains comprises a variable domain (VL) comprising an arginine substituted at residue 38 (or an arginine at residue 38) and a WT constant domain (C_(L)), wherein the V_(H) domain comprising a lysine substituted at residue 39 (or a lysine at residue 39) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat and the human IgG1 or human IgG4 C_(H)1 domain comprising an alanine substituted at residue 172 (or an alanine at residue 172) and a glycine substituted at residue 174 (or a glycine at residue 174) together with the (V_(L)) comprising an arginine substituted at residue 1 (or an arginine at residue 1) and an aspartic acid substituted at residue 38 (or an aspartic acid at residue 38) and the C_(L) domain comprising a tyrosine substituted at residue 135 (or a tyrosine at residue 135) and a tryptophan substituted at residue 176 (or a tryptophan at residue 176) form a first Fab which directs binding to a first target; and the V_(H) domain comprising a tyrosine substituted at residue 39 (or a tyrosine at residue 39) and the WT human IgG1 or human IgG4 C_(H)1 domain together with the (q) comprising an arginine substituted at residue 38 (or an arginine at residue 38) and the WT C_(L) domain form a second Fab which directs binding to a second target which is different from the first target.

Even more particular, the present invention combines the afore-mentioned C_(H)3 domain designs with Fab designs to provide an IgG bispecific antibody, wherein: (a) one of said first or second heavy chains further comprises a variable domain (V_(H)) comprising a lysine substituted at residue 39 (or a lysine at residue 39) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and a human IgG1 or human IgG4 C_(H)1 domain comprising an alanine substituted at residue 172 (or an alanine at residue 172) and a glycine substituted at residue 174 (or a glycine at residue 174); (b) one of said first or second light chains comprises a kappa variable domain (V_(L)) comprising an arginine substituted at residue 1 (or an arginine at residue 1) and an aspartic acid substituted at residue 38 (or an aspartic acid at residue 38), and a constant domain (C_(L)), comprising a tyrosine substituted at residue 135 (or a tyrosine at residue 135) and a tryptophan substituted at residue 176 (or a tryptophan at residue 176); (c) the other of said first or second heavy chains further comprises a variable domain (V_(H)) comprising a tyrosine substituted at residue 39 (or a tyrosine at residue 39) and an arginine substituted at residue 105 (or an arginine at residue 105) and a human IgG1 or human IgG4 C_(H)1 domain comprising a cysteine substituted at residue 127 (or a cysteine at residue 127), an aspartic acid substituted at residue 228 (or an aspartic acid at residue 228), and a glycine substituted at residue 230 (or a glycine at residue 230); and (d) the other of said first or second light chains comprises a variable domain (VL) comprising an arginine substituted at residue 38 (or an arginine at residue 38) and an aspartic acid substituted at residue 42 (or an aspartic acid at residue 42), and a constant domain (C_(L)) comprising a lysine substituted at residue 122 (or a lysine at residue 122), wherein the V_(H) domain comprising a lysine substituted at residue 39 (or a lysine at residue 39) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat and the human IgG1 or human IgG4 C_(H)1 domain comprising an alanine substituted at residue 172 (or an alanine at residue 172) and a glycine substituted at residue 174 (or a glycine at residue 174) together with the (V_(L)) comprising an arginine substituted at residue 1 (or an arginine at residue 1) and an aspartic acid substituted at residue 38 (or an aspartic acid at residue 38) and the C_(L) domain comprising a tyrosine substituted at residue 135 (or a tyrosine at residue 135) and a tryptophan substituted at residue 176 (or a tryptophan at residue 176) form a first Fab which directs binding to a first target; and the V_(H) domain comprising a tyrosine substituted at residue 39 (or a tyrosine at residue 39) and an arginine substituted at residue 105 (or an arginine at residue 105) and the human IgG1 or human IgG4 C_(H)1 domain comprising a cysteine substituted at residue 127 (or a cysteine at residue 127), an aspartic acid substituted at residue 228 (or an aspartic acid at residue 228), and a glycine substituted at residue 230 (or a glycine at residue 230) together with the (V_(L)) comprising an arginine substituted at residue 38 (or an arginine at residue 38) and an aspartic acid substituted at residue 42 (or an aspartic acid at residue 42) and the C_(L) domain comprising a lysine substituted at residue 122 (or a lysine at residue 122) form a second Fab which directs binding to a second target which is different from the first target.

The present invention also provides processes for preparing the IgG bispecific antibodies of the present invention. In particular, the present invention provides a process for preparing an IgG bispecific antibody, comprising: (1) co-expressing in a host cell: (a) a first nucleic acid sequence encoding a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or human IgG4 constant region, wherein said first human IgG1 or human IgG4 constant region comprises an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) a second nucleic acid sequence encoding a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) a third nucleic acid sequence encoding a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or human IgG4 constant region, wherein said second human IgG1 or human IgG4 constant region comprises a valine or methionine substituted at residue 366 (or a valine or methionine at residue 366) and a valine substituted at residue 409 (or a valine at residue 409) of the CH3 domain; and (d) a fourth nucleic acid sequence encoding a second light chain, wherein said second light chain comprises a second variable domain (VD and a second constant domain (C_(L)), wherein one of said first or second heavy chain variable domains and one of said first or second light chain variable domains each comprise three complementarity determining regions (CDRs) which direct binding to a first antigen, and the other of said first or second heavy chain variable domains and first or second light chain variable domains each comprise three complementarily determining regions (CDRs) which direct binding to a second antigen that differs from said first antigen; (2) cultivating said host cell under conditions such that said first and second heavy chains and said first and second light chains are produced; and (3) recovering from said host cell an IgG bispecific antibody comprising a first and second antigen binding fragment (Fab) wherein said first Fab comprises one of said first or second V_(H) domains and one of said first or second V_(L) domains, each of which comprise three CDRs which direct binding to a first antigen, and said second Fab comprises the other of said first or second V_(H) domains and the other of said first or second V_(L) domains, each of which comprise three CDRs which direct binding to a second antigen that differs from the first antigen.

In a particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or human IgG4 constant region, wherein said first human IgG1 or human IgG4 constant region comprises an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or human IgG4 constant region, wherein said second human IgG1 or human IgG4 constant region comprises a valine substituted at residue 366 (or a valine at residue 366) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or human IgG4 constant region, wherein said first human IgG1 or human IgG4 constant region comprises an alanine substituted at residue 407 (or an alanine at residue 407) and a methionine substituted at residue 399 (or a methionine at residue 399) of the C_(H)3 domain; (b) the second nucleic acid sequence encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid sequence encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (KM and a second human IgG1 or human IgG4 constant region, wherein said second human IgG1 or human IgG4 constant region comprises a valine substituted at residue 366 (or a valine at residue 366) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or human IgG4 constant region, wherein said first human IgG1 or human IgG4 constant region comprises an alanine substituted at residue 407 (or an alanine at residue 407), a methionine substituted at residue 399 (or a methionine at residue 399) and an aspartic acid substituted at residue 360 (or an aspartic acid at residue 360) of the C_(H)3 domain; (b) the second nucleic acid sequence encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid sequence encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or human IgG4 constant region, wherein said second human IgG1 or human IgG4 constant region comprises a valine substituted at residue 366 (or a valine at residue 366), a valine substituted at residue 409 (or a value at residue 409), and an arginine substituted at residues 345 and 347 (or an arginine at residues 345 and 347) of the C_(H)3 domain; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or human IgG4 constant region, wherein said first human IgG1 or human IgG4 constant region comprises an aspartic acid substituted at residue 357 (or an aspartic acid at residue 357), a glutamine substituted at residue 364 (or a glutamine at residue 364) and an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) the second nucleic acid sequence encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid sequence encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or human IgG4 constant region, wherein said second human IgG1 or human IgG4 constant region comprises a serine substituted at residue 349 (or a serine at residue 349), a valine substituted at residue 366 (or a valine at residue 366), a tyrosine substituted at residue 370 (or a tyrosine at residue 370) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequences encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or human IgG4 constant region, wherein said first human IgG1 or human IgG4 constant region comprises an aspartic acid substituted at residue 357 (or an aspartic acid at residue 357), a glutamine substituted at residue 364 (or a glutamine at residue 364) and an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) the second nucleic acid sequences encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid sequences encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain and a second human IgG1 or human IgG4 constant region, wherein said second human IgG1 or human IgG4 constant region comprises a serine substituted at residue 349 (or a serine at residue 349), a methionine substituted at residue 366 (or a methionine at residue 366), a tyrosine substituted at residue 370 (or a tyrosine at residue 370) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) the fourth nucleic acid sequences encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or human IgG4 constant region, wherein said first human IgG1 or human IgG4 constant region comprises an aspartic acid substituted at residue 357 (or an aspartic acid at residue 357), an arginine substituted at residue 364 (or an arginine at residue 364) and an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (h) the second nucleic acid sequence encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid sequence encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or human IgG4 constant region, wherein said second human IgG1 or human IgG4 constant region comprises a serine substituted at residue 349 (or a serine at residue 349), a valine substituted at residue 366 (or a valine at residue 366), a tyrosine substituted at residue 370 (or a tyrosine at residue 370) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In yet another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or human IgG4 constant region, wherein said first human IgG1 or human IgG4 constant region comprises a glycine substituted at residue 356 (or a glycine at residue 356), an aspartic acid substituted at residue 357 (or an aspartic acid at residue 357), a glutamine substituted at residue 364 (or a glutamine at residue 364) and an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) the second nucleic acid sequence encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid sequence encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or human IgG4 constant region, wherein said second human IgG1 or human IgG4 constant region comprises a serine substituted at residue 349 (or a serine at residue 349), a valine substituted at residue 366 (or a valine at residue 366), a tyrosine substituted at residue 370 (or a tyrosine at residue 370) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In yet another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or human IgG4 constant region, wherein said first human IgG1 or human IgG4 constant region comprises a glycine substituted at residue 356 (or a glycine at residue 356), an aspartic acid substituted at residue 357 (or an aspartic acid at residue 357), a glutamine substituted at residue 364 (or a glutamine at residue 364) and an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) the second nucleic acid sequence encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid sequence encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or human IgG4 constant region, wherein said second human IgG1 or human IgG4 constant region comprises a serine substituted at residue 349 (or a serine at residue 349), a methionine substituted at residue 366 (or a methionine at residue 366), a tyrosine substituted at residue 370 (or a tyrosine at residue 370) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In yet another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 or human IgG4 constant region, wherein said first human IgG1 or human IgG4 constant region comprises an aspartic acid substituted at residue 357 (or an aspartic acid at residue 357), an arginine substituted at residue 364 (or an arginine at residue 364) and an alanine substituted at residue 407 (or an alanine at residue 407) of the C_(H)3 domain; (b) the second nucleic acid sequence encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid sequence encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 or human IgG4 constant region, wherein said second human IgG1 or human IgG4 constant region comprises a serine substituted at residue 349 (or a serine at residue 349), a methionine substituted at residue 366 (or a methionine at residue 366), a tyrosine substituted at residue 370 (or a tyrosine at residue 370) and a valine substituted at residue 409 (or a valine at residue 409) of the C_(H)3 domain; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

As particular embodiments to the processes of the present invention, processes for preparing IgG bispecific antibodies are provided, wherein the nucleic acids encoding the first and second heavy chains each encode human IgG1 constant regions comprising C_(H)2-C_(H)3 segments of a particular amino acid sequence. Thus, in a particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:7; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:42; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:38; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:42; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:39; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:43; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In yet another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:40; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:44; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In yet another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ NO:41; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:44; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In still another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:40; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:46; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:40; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:45; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In yet another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:41; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:45; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In a further particular embodiment to the process for preparing an IgG-bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:41; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (q) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG1 constant region, wherein said second human IgG1 constant comprises an amino acid sequence as given by SEQ ID NO:46; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ NO:60; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:65; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:61; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:65; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:62; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:66; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In yet another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:63; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (q) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:67; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In yet another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:64; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:67; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In still another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:63; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:69; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:63; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:68; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In yet another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:64; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:68; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

In a further particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (V_(H)) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:64; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (V_(L)) and a first constant domain (C_(L)); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (V_(H)) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:69; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (V_(L)) and a second constant domain (C_(L)).

As an even more particular embodiment to the processes for preparing an IgG bispecific antibody of the present invention, the present invention further combines C_(H)3 domain designs in the IgG1, IgG2 or IgG4 constant regions with Fab designs as described in Lewis et al. (2014) (and WO2014/150973) in the process. In particular, the present invention provides a process for preparing an IgG bispecific antibody according to any one of the afore-mentioned processes, wherein: (a) one of said first or third nucleic acid sequences encodes a heavy chain further comprising a variable domain (V_(H)) comprising a lysine substituted at residue 39 (or a lysine at residue 39) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and a human IgG1 or human IgG4 C_(H)1 domain comprising an alanine substituted at residue 172 (or an alanine at residue 172) and a glycine substituted at residue 174; (b) one of said second or fourth nucleic acid sequences encodes a light chain comprising a kappa variable domain (V_(L)) comprising an arginine substituted at residue 1 (or an arginine at residue 1) and an aspartic acid substituted at residue 38 (or an aspartic acid at residue 38), and a constant domain (C_(L)) comprising a tyrosine substituted at residue 135 (or a tyrosine at residue 135) and a tryptophan substituted at residue 176 (or a tryptophan at residue 176); (c) the other of said first or third nucleic acid sequences encodes a heavy chain further comprising a variable domain (V_(H)) comprising a tyrosine substituted at residue 39 (or a tyrosine at residue 39) and a WT human IgG1 or human IgG4 C_(H)1 domain; and (d) the other of said second or fourth nucleic acid sequences encodes a light chain comprising a variable domain (V_(L)) comprising an arginine substituted at residue 38 (or an arginine at residue 38) and a WT constant domain (C_(L)), wherein the IgG bispecific antibody recovered comprises: a first Fab comprising (i) the variable domain (V_(H)) comprising a lysine substituted at residue 39 (or a lysine at residue 39) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and the human IgG1 or human IgG4 C_(H)1 domain comprising an alanine substituted at residue 172 (or an alanine at residue 172) and a glycine substituted at residue 174 (or a glycine at residue 174), together with (ii) the light chain comprising a kappa variable domain (V_(L)) comprising an arginine substituted at residue 1 (or an arginine at residue 1) and an aspartic acid substituted at residue 38 (or an aspartic acid at residue 38), and a constant domain (C_(L)) comprising a tyrosine substituted at residue 135 (or a tyrosine at residue 135) and a tryptophan substituted at residue 176 (or a tryptophan at residue 176); and a second Fab comprising (i) the variable domain (V_(H)) comprising a tyrosine substituted at residue 39 (or a tyrosine at residue 39) and a WT human IgG1 or human IgG4 C_(H)1 domain, together with (ii) the variable domain (V_(L)) comprising an arginine substituted at residue 38 (or an arginine at residue 38) and a WT constant domain (C_(L)).

Even more particular to the processes for preparing an IgG bispecific antibody of the present invention, the present invention combines the afore-mentioned C_(H)3 domain designs with additional Fab designs in the process. Thus, the present invention provides a process for preparing an IgG bispecific antibody according to any one of the afore-mentioned processes, wherein: (a) one of said first or third nucleic acid sequences encodes a heavy chain further comprising a variable domain (V_(H)) comprising a lysine substituted at residue 39 (or a lysine at residue 39) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and a human IgG1 or human IgG4 C_(H)1 domain comprising an alanine substituted at residue 172 (or an alanine at residue 172) and a glycine substituted at residue 174 (or a glycine at residue 174); (b) one of said second or fourth nucleic acid sequences encodes a light chain comprising a kappa variable domain (V_(L)) comprising an arginine substituted at residue 1 (or an arginine at residue 1) and an aspartic acid substituted at residue 38 (or an aspartica acid at residue 38), and a constant domain (C_(L)) comprising a tyrosine substituted at residue 135 (or a tyrosine at residue 135) and a tryptophan substituted at residue 176 (or a tryptophan at residue 176); (c) the other of said first or third nucleic acid sequences encodes a heavy chain further comprising a variable domain (V_(H)) comprising a tyrosine substituted at residue 39 (or tyrosine at residue 39) and an arginine substituted at residue 105 (or an arginine at residue 105) and a human IgG1 or human IgG4 C_(H)1 domain comprising a cysteine substituted at residue 127 (or a cysteine at residue 127), an aspartic acid substituted at residue 228 (or an aspartic acid at residue 228), and a glycine substituted at residue 230 (or a glycine at residue 230); and (d) the other of second or fourth nucleic acid sequences encodes a light chain comprising a variable domain (VL) comprising an arginine substituted at residue 38 (or an arginine at residue 38) and an aspartic acid substituted at residue 42 (or an aspartic acid at residue 42), and a constant domain (C_(L)) comprising a lysine substituted at residue 122 (or a lysine at residue 122), wherein the IgG bispecific antibody recovered comprises: a first Fab comprising (i) the variable domain (V_(H)) comprising a lysine substituted at residue 39 (or a lysine at residue 39) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and the human IgG1 or human IgG4 C_(H)1 domain comprising an alanine substituted at residue 172 (or an alanine at residue 172) and a glycine substituted at residue 174 (or a glycine at residue 174), together with (ii) the light chain comprising a kappa variable domain (V_(L)) comprising an arginine substituted at residue 1 (or an arginine at residue 1) and an aspartic acid substituted at residue 38 (or an aspartic acid at residue 38), and a constant domain (C_(L)) comprising a tyrosine substituted at residue 135 (or a tyrosine at residue 135) and a tryptophan substituted at residue 176 (or a tryptophan at residue 176); and a second Fab comprising (i) the variable domain (V_(H)) comprising a tyrosine substituted at residue 39 (or a tyrosine at residue 39) and an arginine substituted at residue 105 (or an arginine at residue 105) and a human IgG1 or human IgG4 C_(H)1 domain comprising a cysteine substituted at residue 127 (or a cysteine at residue 127), an aspartic acid substituted at residue 228 (or an aspartic acid at residue and a glycine substituted at residue 230 (or a glycine at residue 230), together with (ii) the variable domain (V_(L)) comprising an arginine substituted at residue 38 (or an arginine at residue 38) and an aspartic acid substituted at residue 42 (or an aspartic acid at residue 42), and a constant domain (C_(L)) comprising a lysine substituted at residue 122 (or a lysine at residue 122).

The present invention further provides an IgG bispecific antibody produced accord to any one of the processes of the present invention.

In addition to the preparation of Fully IgG BsAbs, the methods described herein may also be employed in the preparation of other multi- or mono-valent antigen binding compounds. FIG. 1, included herein, provides a schematic diagram of a Fully IgG BsAb, as well as other antigen binding compounds that one of skill in the art could prepare using the C_(H)3 domain designs, or the C_(H)3 domain designs plus Fab designs, as described herein.

The present invention further provides nucleic acid sequences encoding the first and second heavy chains and the first and second light chains of any of the IgG BsAbs of the present invention. In addition, the present invention also provides vectors comprising nucleic acid sequences encoding the first heavy chain, the first light chain, the second heavy chain and/or the second light chain of any of the IgG BsAbs of the present invention. Further still, the present invention provides host cells comprising nucleic acid sequences encoding the first heavy chain, the first light chain, the second heavy chain and the second light chain of any of the IgG BsAbs of the present invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A-1F provides a schematic diagram of antigen binding compounds that may be prepared using the CH3 domain designs, methods or procedures of the present invention, including Fully IgG BsAbs (A), One-arm antibodies (B), a Tandem Fab-Fc molecules (C), IgG-scFv-Fc molecules (D), and IgG-scFv molecules (E and F). Detailed Legend:

FIG. 1A. Fully IgG BsAb comprising two Fabs (1 and 2) to separate targets and an Fc.

FIG. 1B. One-arm antibody comprising one Fab and one Fc.

FIG. 1C. Tandem Fab-Fc comprising two Fabs (1 and 2) to separate targets linked head-to-tail, and one Fc.

FIG. 1D. IgG-scFv-Fc comprising one Fab (to a first target) and an scFv (to a second target) and one Fc.

FIG. 1E. IgG-scFv comprising two Fabs (1 and 2) to separate targets, a scFv to a third target, and an Fc.

FIG. 1F. IgG-scFv comprising two Fabs (1 and 2) to separate targets, two scFvs (1 and 2) to separate targets, and an Fc.

“Fc(A)” and “Fc(B)” represent distinct CH2-CH3 segments containing CH3 designs and which form the Fc heterodimer.

The general structure of an “IgG antibody” is very well-known. A wild type (WT) antibody of the IgG type is hetero-tetramer of four polypeptide chains (two identical “heavy” chains and two identical “light” chains) that are cross-linked via intra- and inter-chain disulfide bonds. Each heavy chain (HC) is comprised of an N-terminal heavy chain variable region (“V_(H)”) and a heavy chain constant region. The heavy chain constant region is comprised of three domains (C_(H)1, C_(H)2, and C_(H)3) as well as a hinge region (“hinge”) between the C_(H)1 and C_(H)2 domains. Each light chain (LC) is comprised of an N-terminal light chain variable region (“V_(L)”) and a light chain constant region (“C_(L)”). The V_(L) and C_(L) regions may be of the kappa (“κ”) or lambda (“λ”) isotypes. Each heavy chain associates with one light chain via an interface between the heavy chain V_(H)-C_(H)1 segment and the light chain V_(L)-C_(L) segment. The association of each V_(H)-C_(H)1/V_(L)-C_(L) forms two identical antigen binding fragments (Fabs) which direct antibody binding to the same target or epitope. Each heavy chain associates with the other heavy chain via an interface between the hinge-C_(H)2-C_(H)3 segments of each heavy chain, with the association between the C_(H)2-C_(H)3 segments forming the Fc region of the antibody. Together, each Fab and the Fc form the characteristic “Y-shaped” architecture of IgG antibodies, with each Fab representing the “arms” of the “Y.” IgG antibodies can be further divided into subtypes, e.g., IgG1, IgG2, IgG3, and IgG4 which differ by the length of the hinge regions, the number and location of inter- and intra-chain disulfide bonds and the amino acid sequences of the respective HC constant regions.

The variable regions of each heavy chain-light chain pair associate to form binding sites. The heavy chain variable region (V_(H)) and the light chain variable region (V_(L)) can be subdivided into regions of hypervariability, termed complementarity determining regions (“CDRs”), interspersed with regions that are more conserved, termed framework regions (“FR”). Each V_(H) and V_(L) is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. CDRs of the heavy chain may be referred to as “CDRH1, CDRH2, and CDRH3” and the 3 CDRs of the light chain may be referred to as “CDRL1, CDRL2 and CDRL3.” The FRs of the heavy chain may be referred to as HFR1, HFR2, HFR3 and HFR4 whereas the FRs of the light chain may be referred to as LFR1, LFR2, LFR3 and LFR4. The CDRs contain most of the residues which form specific interactions with the antigen.

As used herein, the terms “IgG bispecific antibody”, “IgG BsAb”, “fully IgG bispecific antibody” or “fully IgG BsAb” refer to an antibody of the typical IgG architecture comprising two distinct Fabs, each of which direct binding to a separate antigen (e.g., different target proteins or different epitopes on the same target protein), and composed of two distinct IgG heavy chains and two distinct light chains. The V_(H)-C_(H)1 segment of one heavy chain associates with the V_(L)-C_(L), segment of one light chain to form a “first” Fab, wherein the V_(H) and V_(L) domains each comprise 3 CDRs which direct binding to a first antigen. The V_(H)-C_(H)1 segment of the other heavy chain associates with the V_(L)-C_(L) segment of the other light chain to term a “second” Fab, wherein the V_(H) and V_(L) domains each comprise 3 CDRs which direct binding to a second antigen that is different than the first. More particularly, the terms “IgG bispecific antibody”, “IgG BsAb”, “fully IgG bispecific antibody” or “fully IgG BsAb” refer to antibodies wherein the HC constant regions are composed of C_(H)1, C_(H)2, and C_(H)3 domains of the IgG1, IgG2 or IgG4 subtype, and particularly the human IgG1, human IgG2 or human IgG4. Even more particular, the terms refer to antibodies wherein the HC constant regions are composed of C_(H)1, C_(H)2, and C_(H)3 domains of the IgG1 or IgG4 subtype, and most particularly the human IgG1 or human IgG4 subtype. In addition, as used herein, the terms “IgG bispecific antibody”, “IgG BsAb”, “fully IgG bispecific antibody” and “fully IgG BsAb” refer to an antibody wherein the constant regions of each HC of the antibody are of the same subtype (for example, each HC of the antibody has C_(H)1, C_(H)2, and C_(H)3 domains of the human IgG1 subtype, or each HC of the antibody has C_(H)1, C_(H)2, and C_(H)3 domains of the human IgG2 subtype, or each HC of the antibody has C_(H)1, C_(H)2, and C_(H)3 domains of the human IgG4 subtype.)

The processes and compounds of the present invention comprise designed amino acid modifications at particular residues within the constant and variable regions of heavy chain and light chain polypeptides. As one of ordinary skill in the art will appreciate, various numbering conventions may be employed for designating particular amino acid residues within IgG constant and variable region sequences. Commonly used numbering conventions include the “Kabat Numbering” and “EU Index Numbering” systems. “Kabat Numbering” or “Kabat Numbering system”, as used herein, refers to the numbering system devised and set forth by the authors in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed, Public Health Service, National institutes of Health, Bethesda, Md. (1991) for designating amino acid residues in both variable and constant domains of antibody heavy chains and light chains. “EU Index Numbering” or “EU Index Numbering system”, as used herein, refers to the numbering convention for designating amino acid residues in antibody heavy chain constant domains, and is also set forth in Kabat et al. (1991). Other conventions that include corrections or alternate numbering systems for variable domains include Chothia (Chothia C, Lesk A M (1987), J Mol Biol 196: 901-917; Chothia, et al. (1989), Nature 342; 877-883), IMGT (Lefranc, et al. (2003), Dev Comp Immunol 27: 55-77), and AHo (Honegger A, Pluckthun A (2001) J Mol Biol 309: 657-670). These references provide amino acid sequence numbering schemes for immunoglobulin variable regions that define the location of variable region amino acid residues of antibody sequences. Unless otherwise expressly stated herein, all references to immunoglobulin heavy chain variable region (i.e., V_(H)), constant region C_(H)1 and hinge amino acid residues (i.e. numbers) appearing in the Examples and Claims are based on the Kabat Numbering system, as are all references to the light chain V_(L) and C_(L) residues. All references to immunoglobulin heavy chain constant regions C_(H)2 and C_(H)3 are based on the EU Index Numbering system. With knowledge of the residue number according to Kabat Numbering or EU Index Numbering, one of ordinary skill can apply the teachings of the art to identify amino acid sequence modifications within the present invention, according to any commonly used numbering convention. Note, while the Examples and Claims of the present invention employ Kabat Numbering or EU Index Numbering to identify particular amino acid residues, it is understood that the SEQ ID NOs appearing in the Sequence Listing accompanying the present application, as generated by Patent In Version 3.5, provide sequential numbering of amino acids within a given polypeptide and, thus, do not conform to the corresponding amino acid residue numbers as provided by Kabat Numbering or EU Index Numbering.

However, as one of skill in the art will also appreciate, CDR sequence length may vary between individual IgG molecules and, further, the numbering of individual residues within a CDR may vary depending on the numbering convention applied. Thus, to reduce ambiguity in the designation of amino acid residues within CDRs, the disclosure of the present invention first employs Kabat Numbering to identify the N-terminal (first) amino acid of the HFR3. The amino acid residue to be modified is then designated as being four (4) amino acid residues upstream (i.e. in the N-terminal direction) from the first amino acid in the reference HFR3. For example, a Fab design used in combination with the C_(H)3 domain designs of the present invention comprises the replacement of a WT amino acid in HCDR2 with a glutamic acid (E) (i.e., Fab Design AB2133(a) comprising R62E mutation). This replacement is made at the residue located four amino acids upstream of the first amino acid of HFR3, according to Kabat Numbering. In the Kabat Numbering system, amino acid residue X66 is the most N-terminal (first) amino acid residue of variable region heavy chain framework three (HFR3). One of ordinary skill can employ such a strategy to identify the first amino acid residue (most N-terminal) of heavy chain framework three (HFR3) from any human IgG1 or IgG4 variable region. Once this landmark is identified, one can then locate the amino acid four residues upstream (N-terminal) to this location and replace that amino acid residue (using standard insertion/deletion methods) with a glutamic acid (E) to achieve the design modification of the invention. Given any variable IgG1 or IgG4 immunoglobulin heavy chain amino acid query sequence of interest to use in the processes of the invention, one of ordinary skill in the art of antibody engineering would be able to locate the N-terminal HFR3 residue in said query sequence and then count four amino acid residues upstream therefrom to arrive at the location in HCDR2 that should be modified to glutamic acid (E).

As used herein, the phrase “ . . . a/an [amino acid name] substituted at residue . . . ”, in reference to a heavy chain or light chain polypeptide, refers to substitution of the parental amino acid with the indicated amino acid. For example, a heavy chain comprising “a lysine substituted at residue 39” refers to a heavy chain wherein the parental amino acid sequence has been mutated to contain a lysine at residue number 39 in place of the parental amino acid. Such mutations may also be represented by denoting a particular amino acid residue number, preceded by the parental amino acid and followed by the replacement amino acid. For example, “Q39K” refers to a replacement of a glutamine at residue 39 with a lysine. Similarly, “39K” refers to replacement of a parental amino acid with a lysine. One of skill in the art will appreciate, however, that as a result of the HC-HC interface design modifications of the present invention, fully IgG BsAbs (and processes for their preparation) are provided wherein the component HC amino acid sequences, or component HC and LC amino acid sequences, comprise the resulting or “replacement” amino acid at the designated residue. Thus, tier example, a heavy chain which “comprises a lysine substituted at residue 39” may alternatively be denoted as a heavy chain “comprising a lysine at residue 39.”

An IgG BsAb of the present invention may be derived from a single copy or clone (e.g. a monoclonal IgG BsAb antibody.) Preferably, an IgG BsAb of the present invention exists in a homogeneous or substantially homogeneous population. In an embodiment, the IgG BsAb, or a nucleic acid encoding a component polypeptide sequence of the IgG BsAb, is provided in “isolated” form. As used herein, the term “isolated” refers to a protein, polypeptide or nucleic acid which is free or substantially free from other macromolecular species found in a cellular environment.

An IgG BsAb of the present invention can be produced using techniques well known in the art, such as recombinant expression in mammalian or yeast cells. In particular, the methods and procedures of the Examples herein may be readily employed. In addition, the IgG BsAbs of the present invention may be further engineered to comprise framework regions derived from fully human frameworks. A variety of different human framework sequences may be used in carrying out embodiments of the present invention. Preferably, the framework regions employed in the processes of the present invention, as well as IgG BsAbs of the present invention are of human origin or are substantially human (at least 95%, 97% or 99% of human origin.) The sequences of framework regions of human origin are known in the art and may be obtained from The Immunoglobulin Factsbook, by Marie-Paule Lefranc, Gerard Lefranc, Academic Press 2001, ISBN 012441351.

Expression vectors capable of directing expression of genes to which they are operably linked are well known in the art. Expression vectors contain appropriate control sequences such as promoter sequences and replication initiation sites. They may also encode suitable selection markers as well as signal peptides that facilitate secretion of the desired polypeptide product(s) from a host cell. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. Nucleic acids encoding desired polypeptides, for example the components of the IgG BsAbs prepared according to the processes of the present invention, may be expressed independently using different promoters to which they are operably linked in a single vector or, alternatively, the nucleic acids encoding the desired products may be expressed independently using different promoters to which they are operably linked in separate vectors. In addition, nucleic acids encoding a particular HC/LC pair of the IgG BsAbs of the present invention may expressed from a first vector, while the other HC/LC pair is expressed from a second vector. Single expression vectors encoding both HC and both LC components of the IgG BsAbs of the present invention may be prepared using standard methods. For example, a pE vector encoding a particular HC/LC pair may be engineered to contain a NaeI site 5 prime of a unique SalI site, outside of the HC/LC expression cassette. The vector may then be modified to contain an AscI site 5 prime of the SalI site using standard techniques. For example, the NaeI to SalI region may be PCR amplified using a 3′ primer containing the AscI site adjacent to the SalI site, and the resulting fragment cloned into the recipient pE vector. The expression cassette encoding a second HC/LC pair, may then be isolated from a second (donor) vector by digesting the vector at suitable restriction sites. For example, the donor vector may be engineered with MluI and SalI sites to permit isolation of the second expression cassette. This cassette may then be ligated into the recipient vector previously digested at the AscI and SalI sites (as AscI and MluI restriction sites have the same overlapping ends.)

As used herein, a “host cell” refers to a cell that is stably or transiently transfected, transformed, transduced or infected with nucleotide sequences encoding a desired polypeptide product or products. Creation and isolation of host cell lines producing an IgG BsAb of the present invention can be accomplished using standard techniques known in the art.

Mammalian cells are preferred host cells for expression of the IgG BsAb compounds according to the present invention. Particular mammalian cells include HEK293, NS0, DG-44, and CHO cells. Preferably, assembled proteins are secreted into the medium in which the host cells are cultured, from which the proteins can be recovered and isolated. Medium into which a protein has been secreted may be purified by conventional techniques. For example, the medium may be applied to and eluted from a Protein A or G column using conventional methods. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, hydroxyapatite or mixed modal chromatography. Recovered products may be immediately frozen, for example at −70° C., or may be lyophilized. As one of skill in the art will appreciate, when expressed in certain biological systems, e.g. mammalian cell lines, antibodies are glycosylated in the Fc region unless mutations are introduced in the Fc to reduce glycosylation. In addition, antibodies may be glycosylated at other positions as well.

The following Examples further illustrate the invention and provide typical methods and procedures for carrying out various particular embodiments of the present invention. However, it is understood that the Examples are set forth the by way of illustration and not limitation, and that various modifications may be made by one of ordinary skill in the art.

Example 1 Computational and Rational Design of CH3/CH3 Interface Modifications

Residues for initial modification at the symmetric C_(H)3/C_(H)3 dimer interface (i.e., Chain A C_(H)3 domain and Chain B C_(H)3 domain) were selected using a combination of computational and rational design strategies. First, using a crystal structure of the human IgG1 C_(H)2-C_(H)3 domains (PDB 1L6X), trimmed of carbohydrate moieties that connect the C_(H)2 domains, the Rosetta software suite and related modeling applications were employed to computationally identify potential modifications that favor heterodimer (i.e., Chain A/Chain B) formation over homodimer (i.e., Chain A/Chain A or Chain B/ChainB) formation. (See, Kaufmann et al. (2010), Biochemistry 49; 2987-2998; Leaver-Fay et al. (2011), Methods Enzymol. 487; 545-574; Kuhlman et al. (2003), Science 302(5649); 1364-1368; and Leaver-Fay et al. (2011), PLos ONE 6(7): e20937; Lewis et al. (2014) Nature Biotech., 32(2)). More than sixty discrete initial designs, falling into varying design paradigms, different amino acid substitutions and/or different amino acid residue positions) were identified, synthesized and tested for heterodimer formation and thermostability (as measured by UPLC, FRET and DSC, as described below.) Select Chain A/Chain B mutation pairs were further optimized rationally and/or assessed for compatibility for combination, including inverted combinations where mutations in one chain of a discrete design pair (e.g. Chain B) are added to the mutations in the opposite chain (e.g. Chain A) of a separate discrete design pair. Optimized and/or combination designs were then assessed computationally and those exhibiting promising heterodimer formation potential, and destabilized homodimer potential, were also synthesized and tested for heterodimer formation (as measured by UPLC and FRET) and thermostability (as measured by DSC.)

A. Computational Modeling

Briefly, Rosetta's multistate design module explores sequence space and for each sequence calculates an energy for each of several “states” based on a weighted sum of energy potentials treating phenomena such as van der Waals forces and hydrogen bonding forces, and then aggregates these energies to compute a fitness for that sequence. The states represent different combinations and conformations of protein chain species, e.g. different conformations of the Chain A/Chain B heterodimer, or different conformations of the Chain A/Chain A or Chain B/Chain B homodimer. The summations of the energy potentials are measured in units known as the Rosetta Energy Unit (REU). These values are interpreted as free energies, but do not directly translate into typical units of energy. Binding energies are computed as the difference between the energy of the bound complex and the energies of its separated components. Using a fitness function which favors the binding and stability of Chain A C_(H)3/Chain B C_(H)3 heterodimers and disfavors the binding of C_(H)3/C_(H)3 homodimers (Chain A/Chain A or Chain B/Chain B), initial sequences for modification are identified.

The identified mutations are subjected to computational docking of the heterodimer and homodimer complexes using RosettaDock via RosettaScripts (see, Chaudhury et al. (2011), PLoS ONE 6(8): DOI: 10.1371journal.pone.0022477 and Fleishman et al. (2011), PLoS ONE 6(6): DOI: 10.1371/journal.pone.0020161). This docking step allows the complexes to relax into more favorable conformations for their sequences and facilitates the comparison of binding energies for the homodimer and heterodimer complexes. Energies are calculated using a variation on Rosetta's standard score function, “Talaris2013” (O'Meara et al. (2015) J. Chem. Theory Comput. 11(2); 609-622), where the atomic-interaction distance was extended to 9 Å and the amino-acid specific reference energies had been refit using Rosetta's automated refitting procedure, OptE (Leaver-Fay et al. (2013) Methods Enzymol. 523; 109-143). Following docking, binding energies are calculated as the difference in energies between the complex and the sidechain-optimized, separated conformations as reported by Rosetta's InterfaceAnalyzer tool (see, Lewis, S. M. and Kuhlman, B. A. (2011), PLoS One 6(6): DOI: 10.1371/journal.pone.0020872). The conformations resulting from the docking simulations of homodimers which display favorable binding energies are used as additional states in subsequent multistate design simulations to further guide the those simulations away from sequences which favor homodimer formation. The multistate-design-followed-by-redocking process is iterated until the binding energies calculated by multistate design match well with binding energies calculated following docking.

Select designs, including further optimized and/or combination designs, resulting from this iterative process and their calculated binding energies are provided in Table 1.

TABLE 1 Rosetta multi-state computational design results. Chain A HC Chain B HC A/B^(e) A/A^(e) B/B^(e) Design C_(H)3 Domain C_(H)3 Domain binding binding binding Construct Mutations^(a) Mutations^(a) energy energy energy WT^(b) None — — −12 — 7.4 Y407A T366V −16.3 −12.5 −11.2 K409V 7.8 Y407A T366V −17.2 −11.3 −5.3 D399M K409V 7.8.60^(b) K360D E345R −51.5 −42.7 −45.0 D399M Q347R Y407A T366V K409V 11.2a^(c) Y349A E357D −18.1 −19.6 −2.2 K370Y S364Q 20.8^(d) Y349S E357D −29.7 −13.4 −14.9 T366V S364Q K370Y Y407A K409V ^(a)Mutations are designated by first identifying the one letter abbreviation for the parental amino acid, the amino acid residue number and the one letter abbreviation for the replacement amino acid. For example, Y407A indicates that residue 407 of is modified from a tyrosine (Y) to an alanine (A). Binding energies were calculated following a fixed-backbone, rigid-body docking protocol, starting from the PDB ID 1L6X crystal structure. ^(b)Binding energies calculated after flexible-backbone relaxation protocol. The addition of backbone flexibility lowers apparent binding energies considerably. ^(c)In the constructs prepared in Section B below, 349A is modified to 349S in HC A as the Y349S mutation was observed in the calculations to make an additional hydrogen bond to 357D in HC B. This modification to Design 11.2a is denoted as Design 11.2. ^(d)Binding energies were calculated following a fixed-backbone, rigid-body docking protocol, starting from a crystal structure of design 11.2. ^(e)The designations “A/B”, “A/A” and “B/B” refer to the CH3 domain hetero- or homodimer chain pairs

B. Design Construct Heterodimer Formation and Thermostability Assessment of Heterodimer Formation by Ultra-Performance Liquid Chromatography (UPLC) Analytical Sizing

To assess the heterodimer formation potential of Chain A C_(H)3 domain/Chain B C_(H)3 domain design pairs, “one-arm” antibody constructs incorporating design modifications in the C_(H)3/C_(H)3 dimer interface are prepared and tested. Unless otherwise indicated, “Chain A” of each construct contains a full heavy chain sequence (with or without C_(H)3 domain design modifications) and “Chain B” of each construct contains an Fc only portion (C_(H)2-C_(H)3 segment plus HA tag) of the heavy chain (with or without CH3 domain design modifications.)

Molecular Biology: Variable heavy domain (V_(H)) and variable light domain (V_(L)) sequences of the anti-cMet clone 5D5 (see U.S. Pat. No. 7,892,550) are synthesized. The V_(H) domain-encoding sequence is cloned into a plasmid (pcDNA3.1(+) (Life Technologies)) containing sequences encoding a mouse kappa chain leader sequence and a complete human IgG1 heavy chain using HindIII/EcoR1 restriction sites. The V_(L) domain-encoding sequence is cloned into a pEHK mammalian expression vector (Lonza) containing a sequence encoding a mouse kappa chain leader sequence and a 3′ kappa constant domain, using the BamHI and EcoRI restriction sites. An HA-tagged Fc construct, to provide the other member of the C_(H)3 dimer interface, is constructed by first PCR amplifying a human IgG1 Fc from a full heavy chain using a forward primer which introduced an HA tag plus a four residue linker at the N-terminus of the chain. The HA-tagged Fc-encoding construct is then cloned into a pcDNA3.1(+) plasmid containing a sequence encoding a mouse kappa chain leader sequence using the BamH1 and EcoR1 restriction sites. Nucleic acid sequence modifications encoding the C_(H)3 domain design pair mutations are introduced using methods known in the art such as Kunkel mutagenesis (See Kunkel (1985) Proc Natl Acad Sci; 82(2):488-492), QUIKCHANGE® site-directed mutagenesis (Agilent), or direct GBLOCK® gene cloning (Integrated DNA Technologies®, IDT) using restriction site cloning using EcoRI and an internal SacII site within the C_(H)2 domain. Mutant C_(H)3 designs were introduced by Kunkel mutagenesis (See Kunkel (1985) Proc Natl Acad Sci; 82(2):488-492), QUIKCHANGE® site-directed mutagenesis (Agilent), or direct cloning from FRET constructs containing C_(H)3 mutations, using restriction site cloning with EcoRI and an internal SacII site within the C_(H)2 domain. The parental protein sequences for the one-arm antibody constructs, prior to incorporation of the C_(H)3 domain design pair modifications (i.e., WT C_(H)3 domains), are provided in SEQ ID NOs: 1-3.

The three plasmids (0.25 μg anti-c-Met VH-human IgG1 HC (with or without C_(H)3 modifications), 0.25 μg HA-tagged human IgG1 Fc portion (with or without C_(H)3 modifications), and 1.5 μg anti-c-Met light chain) are transiently transfected into 2 mL of HEK293F cells. Transfected cells are grown at 37° C. in a 5% CO₂ incubator while shaking at 125 rpm for 5 days. Secreted protein is harvested by centrifugation at 2K rpm for 5 min. and recovery of the supernatant. Expressed protein is purified from the supernatant using PureProteome Protein G Magnetic Beads (EMD Millipore), a DynaMag Magnetic Particle Concentrator (Invitrogen), and Protein G wash and Elution Buffers (Biomiga), as per manufacturer instructions. Eluted samples are neutralized with 1M Tris pH9.0 (Sigma) and filtered with an Ultrafree-MC-GV centrifugal filter (Millipore), per manufacturer instructions.

UPLC Detection: 30 μl samples of expressed protein are added to Waters UPLC tubes, from which 10 μl is injected into a Waters Acquity UPLC with a BEH200 SEC column, equilibrated in PBS and run at 0.3 ml/min. A dilution series of purified MetMab is also run as a standard. Resulting A280 chromatogram peaks from the UPLC traces are deconvoluted and integrated using a custom set of GNU Octave scripts to quantify % heterodimerization by peak area. Tables 2 and 3 below provide heterodimer formation data, as determined by UPLC, for select designs, including further optimized or combination designs. The following provides experimental details of the treatment of the UPLC traces, including various characteristic peaks obtained, as well as procedures employed for curve fitting and data interpretation.

From run to run, the retention times for the various protein species may shift forward and backwards in time together so that if a Chain A HC/Chain B HC heterodimer were to elute at 4.15 m, then the Chain A/Chain A homodimer would elute at 3.8 m, but if the Chain A HC/Chain B HC heterodimer were to elute, for example, 0.35 minutes later at 4.5 m, then the Chain A HC/Chain A. HC homodimer would elute similarly later at 4.1.5 m. A sharp peak between 5.5 and 6.5 m, from a non-antibody species, is characteristic of the UPLC traces, with no recorded species appearing 0.25 m before the peak. A linear baseline absorption is subtracted from all of the UPLC traces. The linear baseline is fit from two points taken as the average absorption between 2.0 m and 2.083 m and the average absorption between 0.25 m and 0.25 m+0.083 m, before the characteristic non-antibody peak at about 6 m. Parameters for the Generalized Exponentially Modified Gaussian (GEMG) curve (Nikitas et al. (2001) J. Chromatog. A, 912: 13-29) are fit for each of the protein species' peaks observed in the traces using data where these peaks are cleanly observed.

The five parameters that describe the shape of the GEMG curves for each of the various species observed in the UPLC traces were fit using traces that unambiguously displayed those species, and then used as seed values for subsequent curve fittings. After the shapes of each of the species were fit, the remaining curves were fit automatically in Octave by scanning the data for peaks and attempting to place the Chain A HC/Chain B HC heterodimer peak in each one and shifting the other peaks with it, miming Octave's fminunc routine to minimize the restrained sum-of-square residuals (SSR), which includes a restraint score on the GEMG-parameter deviations, and then, following optimization, picking the Chain A HC/Chain B HC heterodimer peak assignment that yields the best SSR. For many curves, however, the best SSR does not represent a reasonable interpretation of the data, and so the peak-placement of the Chain A HC/Chain B HC heterodimer is manually determined. The volumes for the peaks are integrated numerically and the molar percentages are then determined by correcting for the absorbance of each species. Otherwise, higher molecular weight contaminants will appear more prominent and lower molecular weight contaminants less prominent than they actually are on a molar basis.

Assessment of Heterodimer Formation by Fluorescence Resonance Energy Transfer (FRET)

To further assess the heterodimer formation potential of Chain A C_(H)3 domain/Chain B C_(H)3 domain design pairs, further constructs incorporating design modifications in the C_(H)3/C_(H)3 dimer interface are prepared and tested, as described below.

Molecular Biology: The construction of vectors housing oligonucleotide sequences used to express proteins for FRET analysis is performed as follows. Annealed oligos (IDT) are is used to introduce a Myc tag into an in-house vector containing a nucleic acid encoding the mouse kappa leader sequence and a wild type human IgG1 Fc. Two complementary Myc oligos that leave protruding 5′ or 3′ overhangs for ligation into a vector cut with the appropriate enzymes are designed. The oligos are annealed and ligated into the in-house vector containing the human IgG1 Fc-encoding sequence digested with the appropriate restriction enzymes. After sequence verification, the vector is digested with appropriate enzymes to then introduce the human EGFR Domain 3 (hEGFR D3)- or mouse VEGFR1 Domain 3 (mVEGFR D3)-encoding sequences.

The hEGFR D3 construct was designed using the crystal structures of the extracellular domains of hEGFR bound to cetuximab (PDB ID 1YY9, Structural basis for inhibition of the epidermal growth factor receptor by cetuximab (Li et al. (2005) Cancer Cell 7: 301-311)) and the D3 domain, specifically, bound to matuzumab (PDB ID 3C09, Matuzumab binding to EGFR prevents the conformational rearrangement required for dimerization. (Holzel et al. (2008) Cancer Cell 13: 365-373)). The hEGFR D3 nucleic acid construct was designed with appropriate restriction sites to enable cloning and was synthesized (IDT®). The hEGFR D3 nucleic acid construct is restriction digested, separated on a 1% agarose gel and the DNA fragment is purified using a Gel Extraction Kit (Qiagen). The purified DNA fragment is ligated into the pcDNA 3.1 mammalian expression plasmid (Life Technologies) between the Myc tag and the human IgG1 Fc, respectively. The construct is then sequence verified for use in subsequent cloning of CH3 designs. A two amino acid, GS, linker is inserted between the EGFR or VEGFR1 D3 domains and the human IgG1-Fc.

The mVEGFR1 D3-encoding sequence is obtained from an in-house source and used as a PCR template. The mVEGFR1 D3 protein binds an in-house generated chimeric Mab (as determined by in-house testing). The mVEGFR1 D3-encoding DNA is amplified by PCR using oligonucleotide primers designed to add restriction sites to enable cloning into the vector (pcDNA containing the Myc tag and human IgG1 Fc encoding sequences). The PCR product is digested with the appropriate restriction enzymes and gel purified. The purified DNA PCR product is then ligated into the pcDNA 3.1 between the Myc tag- and the IgG1 Fc-encoding sequences, respectively. The construct is then sequence verified for use in subsequent cloning of C_(H)3 designs.

The sequence of the hEGFR D3-Fc protein containing the wild-type human IgG1 C_(H)3 domain is provided below in SEQ ID. NO. 4. The sequence of the mVEGFR1 D3-Fc protein containing the wild-type human IgG1 C_(H)3 domain is provided below in SEQ ID. NO. 5. Mutant C_(H)3 designs are introduced by direct GBLOCK® gene cloning (IDT®) using restriction sites BsrGI and EcoRI and/or QUIKCHANGE® site-directed mutagenesis (Agilent).

Each plasmid is scaled-up by transformation into TOP10 E. coli, mixed with 100 mL luria broth in a 250 mL baffled flask, and shaken O/N at 220 rpm. Large scale plasmid purifications are performed using the BenchPro 2100 (Life Technologies) or HiSpeed Plasmid Maxi Kit (Qiagen) according to the manufacturer's instructions. For protein production, plasmids harboring the Chain A and Chain B DNA sequences are transfected (1:1 plasmid) into HEK293F cells using Freestyle transfection reagents and protocols provided by the manufacturer (Life Technologies). Transfected cells are grown at 37° C. in a 5% CO2 incubator while shaking at 125 rpm for 5 days. Secreted protein is harvested by centrifugation at 10 K rpm for 5 min. Supernatants are passed through 2 μm filters (both large scale and small scale) for purification.

FRET Detection: The Fab detection reagents for use in the FRET assay are generated as follows. The matuzumab human IgG1 MAb (anti-hEGFR D3) was constructed in-house as described previously (Lewis et al., 2014) and a Fab generated from the matuzumab IgG1 MAb using papain digestion as described previously (Jordan et al. (2009) Proteins 77: 832-41). The anti-mVEGFR1 D3 Fab protein is generated in house from published sequences (WO2014/150314). Fluorescent isothiocyanato-activated Europium-W1024 (Perkin Elmer Life Sciences) labeling of the anti-mVEGFR1 D3 and Matuzumab Fabs is performed according to the manufacturer's instructions. Fluorescent Cy5 (Amersham Pharmacia Biotech) labeling of the anti-mVEGFR1 D3 and Matuzumab Fabs is performed according to the manufacturer's instructions.

To test the C_(H)3 designs in the FRET assay, Europium(Eu)-labeled mVEGFR1. D3 Fab, or Eu-labeled Matuzumab FAb (anti-hEGFR D3), is mixed with Cy5-labeled anti-mVEGFR1 D3, or Cy5-labeled Matuzumab Fab to final concentrations of 1.25 μg/ML Eu-reagent, 2.5 μg/mL Cy5-reagent in diluted HEK293F cell culture supernatants containing secreted protein resulting from co-expression of both EGFR-D3-Fc (Chain A) and VEGFR1-D3-Fc (Chain B). The cell culture supernatants are diluted 1:10 or 1:40 in PBS, 10 mg/mL BSA, 0.1% Tween-20 for 1 mL and 2 mL transient transfections, respectively, prior to the FRET measurements. These particular supernatant dilutions result in a roughly 0.5-1 μg/mL final Protein-Fc concentration optimal for measuring the homodimer/heterodimer ratios. Mixing of the Eu- and Cy5-labeled Matuzumab Fabs enables detection of EGFR-D3-Fc AA homodimer. Mixing Eu- and Cy5-labeled anti-VEGFR1-D3 Fabs enables detection of VEGFR1-D3-Fc BB homodimer. Mixing of Eu-labeled anti-VEGFR1-D3 Fab with Cy5-labeled anti-Cy5-labeled Matuzumab enables detection of EGFR-D3-Fc/VEGFR1-D3-Fc AB heterodimer. The simultaneous binding of Eu-labeled Fab and Cy5-labeled Fab to a single protein molecule (either homodimer-Fc or heterodimer-Fc depending on the Fab combinations) results in a time-resolved fluorescence resonance energy transfer (TR-FRET) from the Europium label to the Cy5 label. 96½ well microtiter plates (black from Costar) containing the diluted supernatants and labeled Fabs are incubated for approximately 30 minutes at room temperature. Fluorescence measurements are carried out on a Wallac Envision 2103 Multilabel Reader with a dual mirror (PerkinElmer Life Sciences) with the laser excitation of the Europium at wavelength at 340 nm and the mission filters Europium 615 and APC 665. Delay between excitation and emission was 20 μs.

Assessment of Heterodimer Thermostability by Differential Scanning Calorimetry (DSC)

Generation of Heterodimeric Fcs Suitable for DSC

To assess the thermostability of Chain A C_(H)3 domain/Chain B C_(H)3 domain design pairs, Fc constructs incorporating design modifications in the C_(H)3/C_(H)3 dimer interface are prepared and tested. To generate Fcs for thermostability analysis, including dimers incorporating C_(H)3 domain design pair mutations, an HA-tagged human IgG1 Fc portion (Chain B C_(H)2-C_(H)3 segment) and a human IgG1 Fc portion without an HA tag (Chain A C_(H)2-C_(H)3 segment) are constructed. The sequence of the HA tagged-human IgG1 Fc portion containing the WT C_(H)3 domain sequence is provided in SEQ ID NO:2. The sequence of the human IgG1 Fc portion (without an HA tag) containing the WT C_(H)3 domain sequence is provided by SEQ ID NO:6.

The C_(H)3 design constructs for use in DSC analysis are made in one of two ways, shuttling from another construct containing a nucleic acid encoding the C_(H)3 design of interest, or site directed mutagenesis. When shuttling between existing constructs, restriction cloning of the C_(H)3 encoding fragment containing the desired mutations is employed. The nucleic acid encoding the C_(H)3 containing the desired mutations is inserted into the vector of interest by digesting both the donor vector and the recipient vector into which the design mutations will be inserted. Both the insert and recipient vector DNA's are purified using gel electrophoresis and the purified insert and receptor vector DNA fragments are then ligated. All ligation constructs are transformed into E. coli strain TOP 10 competent cells (Life Technologies). When site-directed mutagenesis is employed, the basic procedure utilizes a supercoiled double-stranded DNA vector containing the wild-type nucleotide sequence of interest and two synthetic oligonucleotide primers (IDT®) containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during thermal cycling by the DNA polymerase (HotStar HiFidelity Kit, Qiagen Cat.202602). Incorporation of the oligonucleotide primers generates a mutated plasmid. Following temperature cycling, the product is treated with Dpn I enzyme. (New England BioLabs, Cat # R0176) The Dpn I enzyme cleaves only methylated parental DNA. The enzyme digested mutated plasmid is then transformed into E. coli strain TOP 10 competent cells (Life Technologies).

Sequenced plasmids are scaled-up for transfection as described above for the FRET constructs. Plasmids are transfected into 293F using the same protocol as described for the FRET constructs above. Secreted protein is harvested by centrifugation at 10 K rpm for 5 min. Supernatants are passed through 2 μm filters for purification. Purification is performed using protein A chromatography as described by Lewis, et al. (2014), Nat. Biotechnol., 32; 191-198.

Following procedures as described above, C_(H)3 designs are incorporated into the Fc portions containing the WT C_(H)3 domain sequences (SEQ ID NO:2 (Chain B) and SEQ ID NO:6 (Chain A)). Differential scanning calorimetry (DSC) measurements are carried out as generally described in Clark, L. A. et al., 2014 J. Struct. Biol. 185:223-227 with a scan rate of 1.5 deg. C./min. All DSC thermograms are fit using analysis software provided by the manufacturer (GE Healthcare).

Table 2 provides heterodimer formation results (as measured by UPLC and FRET) as well as thermostability data (as determined by DSC) for select designs.

TABLE 2 Heterodimeric potential of computational designs and their thermal stability CH3 Mutations* Total fit Chain Chain % heterodimer area ^(c) % heterodimer T_(m) of CH3 design A B (UPLC) ^(a, b) UPLC (FRET signal)^(d) (DSC) WT 51.3 ± 0.7 (12) 99.5 ± 0.2 56.6 83  7.4 Y407A T366V 86 98 97.5 75 K409V  7.8 Y407A T366V 93 99 85.8 71 D399M K409V 11.2 Y349S E357D 74 99 93.6 70.67 K370Y S364Q *Mutations are designated by first identifying the one letter abbreviation for the parental amino acid, the amino acid residue number and the one letter abbreviation for the replacement amino acid. For example, Y407A indicates that residue 407 of is modified from a tyrosine (Y) to an alanine (A). ^(a) Values represent single UPLC experiments unless numbers appear in parenthesis. Where numbers appear in parenthesis, the values represent the arithmetic mean +/− standard error and the number within the parens represents the number of UPLC experimental repeats. ^(b) UPLC peaks were fit using an automated curve fitting protocol, with molar fractions taken as peak volumes normalized by expected extinction coefficients. Because the two chains might not express at equal levels, monomeric impurities are not included when computing the percentage heterodimer. ^(c) Total fit area indicates how much of the chromatographic trace can be fit by GEMG curves and suggests how much remains as unidentifiable contaminant species. ^(d)This assay should not be considered quantitative. The labeled Fabs and the Fcs are at about equal concentrations, so not all Fcs will be bound to Fabs. When detecting the AA homodimer concentration, some of the anti-EGFR Fabs will be bound to the AB heterodimer, and some AA homodimers will bind to two Eu³⁺-labled Fabs or to two Cy5-labled Fabs and will thus not FRET, or they will bind to only a single Fab and not FRET. The percentages reported are calculated as relative FRET intensities; e.g. % AB = FRET(AB)/(FRET(AA) + FRET(AB) + FRET(BB)).

Table 3 provides additional heterodimer formation results and thermostability data for select initial designs (e.g., Design 7.8) as well as further optimized variants of Design 7.8 and the combination Design 20.8.

TABLE 3 Heterodimeric assembly and thermal stability of computational designs 20.8, 7.8, and optimized variants CH3 Mutations* Chain Chain % heterodimer Total fit % heterodimer Tm of CH3 design A B (UFLC) ^(a, b) area ^(c) (FRET signal)^(d) (DSC) ^(c) WT  51.3 ± 0.7 (n = 12)  99.5 ± 0.2 58 20.8 Y349S E357D 84.9 ± 1.4 (n = 6) 99.0 ± .3 86 69.9, 70.3, K370Y S364Q 69.5, 70.4 T366V Y407A K409V 20.8b E357D Y349S 92.5 ± 0.1 (n = 2) 100.0 ± .2  95 ND S364Q K370Y Y407A T366V K409V 20.8.26 Y349S E357D 86  99 96 K370Y S364Q T366M Y407A K409V 20.8.26b E357D Y349S 93 100 ND ND S364Q K370Y Y407A T366M K409V 20.8.31 Y349S E357D 86.5 ± 3.6 (n = 3) 99.6 ± .3 95 68.9, 69.6 T366V S364R K370Y Y407A K409V 20.8.31b E357D Y349S 85 100 ND ND S364R T366V Y407A K370Y K409V 20.8.33 Y349S E356G 93  99 98 ND T366V E357D K370Y S364Q K409V Y407A 20.8.33b E356G Y349S 92 100 ND ND E357D T366V S364Q K370Y Y407A K409V 20.8.34 Y349S E356G 83.8 ± 3.5 (n = 3) 99.9 ± .4   95.5 68.9 K370Y E357D T366M S364Q K409V Y407A 20.8.34b E356G Y349S 94 100 ND ND E357D K370Y S364Q T366M Y407A K409V 20.8.37 Y349S E357D 83.5 ± 1.7 (n = 2) 100.0 ± .2  ND 68.9 K370Y S364R T366M Y407A K409V 20.8.37b E357D Y349S 85 100 ND ND S364R K370Y Y407A T366M K409V 7.8 Y407A T366V 89.9 ± 1.9 (n = 6) 99.3 ± .3   85.8 71   D399M K409V 7.8b T366V Y407A 88.6 ± 1.0 (n = 6) 99.4 ± .2   92.2 ND K409V D399M 7.8.60 K360D E345R 93  99 ND 70.4 D399M Q347R Y407A T366V K409V 7.8.60b E345R K360D 93.3 ± 1.3 (n = 3) 99.7 ± .1 ND ND Q347R D399M T366V Y407A K409V *Mutations are designated by first identifying the one letter abbreviation for the parental amino acid, the amino acid residue number and the one letter abbreviation for the replacement amino acid. For example, Y407A indicates that residue 407 of is modified from a tyrosine (Y) to an alanine (A). ^(a) Values represent single UPLC experiments unless numbers appear in parenthesis. Where numbers appear in parenthesis, the values represent the arithmetic mean +/− standard error and the number within the parens represents the number of UPLC experimental repeats. ^(b) UPLC peaks were fit using an automated curve fitting protocol, with molar fractions taken as peak volumes normalized by expected extinction coefficients. Because the two chains might not express at equal levels, monomeric impurities are not included when computing the percentage heterodimer. ^(c) Total fit area indicates how much of the chromatographic trace can be fit by GEMG curves and suggests how much remains as unidentifiable contaminant species. ^(d)This assay should not be considered quantitative. The labeled Fabs and the Fcs are at about equal concentrations, so not all Fcs will be bound to Fabs. When detecting the AA homodimer concentration, some of the anti-EGFR Fabs will be bound to the AB heterodimer, and some AA homodimers will bind to two Eu³⁺-labled Fabs or to two Cy5-labled Fabs and will thus not FRET, or they will bind to only a single Fab and not FRET. The percentages reported are calculated as relative FRET intensities; e.g. % AB = FRET(AB)/(FRET(AA) + FRET(AB) + FRET(BB)). ^(e) Multiple values in the DSC column represent values from duplicate experiments.

The data is Tables 2 and 3 demonstrate that exemplified CH3 designs yield significant enhancement of Chain A/Chain B heterodimerization relative to dimer constructs which contain only wild-type CH3 domains. Select heterodimer-favoring designs are incorporated into complete IgG heavy chains and the assembly of particular IgG bispecific antibodies is assessed as described in Example 2, below.

Example 2 Complete IgG Bispecific Antibodies (IgG1 HC Backbones) Comprising C_(H)3/C_(H)3 Interface Modifications

Four published antibodies (with published sequences) were chosen to generate three IgG BsAbs. The first MAb pair to be expressed as an IgG BsAb consists of pertuzumab (anti-HER-2) (see, Nahta, Hung & Esteva (2004), Cancer Res. 64; 2343-2346; and Franklin et al. (2004), Cancer Cell. 5(4); 317-28) and BHA10 (anti-LTβR) (see, Michaelson et al. (2009), mAbs 1; 128-141; and Jordan et al. (2009), Proteins 77(4); 832-41.) The second MAb pair to be expressed as an IgG BsAb consists of a combination of MetMAb (anti-cMET) (see, Jin et al. (2008), Cancer Res. 68; 4360-4368; and U.S. Pat. No. 7,892,550) and matuzumab (anti-EGFR) (see, Bier et al. (1998), Cancer Immunol. Immunother. 46; 167-173; and Schmiedel et al. (2008) Cancer Cell. 13(4); 365-73. doi: 10.1016/j.ccr.2008.02.019.) Lastly, pertuzumab was paired with matuzumab to form a third set of IgG BsAbs. All BsAbs were tested for assembly using select C_(H)3 designs, as described in Example 1, or WT C_(H)3 domain sequences. The C_(H)3 designs are incorporated into the C_(H)3 domains of each parental Mab pair, with each Mab C_(H)3 domain receiving one set of mutations of a particular design pair (i.e, either the A Chain or B Chain mutations), and the other Mab C_(H)3 domain receiving the other set of mutations of the design pair. In addition, each HC and LC prepared and tested included previously described mutations in the Fab region to promote proper HC-LC pairing as well. Matuzumab and BHA10 HCs and LCs contain Design H4WT (+DR_CS), while the pertuzumab and MetMAb HCs and LCs contain Design AB2133(a), each as described in WO2014/150973 (see also, Lewis et al. (2014), Nature Biotech. 32; 191-198 (Designs VRD2, VRD1 and CRD2).

Methods

The plasmids for the IgG BsAbs are obtained in-house (see Lewis et. al (2014) Nat Biotechnol. 32; 191-198). The construction of BsAbs with each set of C_(H)3 designs are done in one of the two following ways.

Oligonucleotide primers with 15 base pair extensions (5′) that are complementary to the N-termini of the V_(H) region (V_(H) forward) and the C-terminus of the C_(H)2 region (C_(H)2 reverse) of the HC are used in a PCR reaction to generate recombinase-compatible inserts of the entire HC except the C_(H)3 domain. A second set of oligonucleotides are used to generate additionally inserts encoding the design-containing C_(H)3 domains. The templates for these additional primers are from the FRET, UPLC or DSC constructs described in Example 1. The 5′ primers for the C_(H)3 domain are complementary to the junction between C_(H)2 and C_(H)3 (called C_(H)2 forward) and the 3′ primers are complementary to the C-terminus of the C_(H)3 region (C_(H)3 reverse). Both the 5′ and 3′ primers contain 15 base pair extensions to allow recombinase-based cloning. The PCR products are gel purified. The BsAb vector(s) are digested with 2 different restriction enzymes, removing the C_(H)1, C_(H)2 and C_(H)3 domains. Recombinase-based cloning is performed using the In-Fusion protocol (Clontech Laboratories, Inc.) to generate the each clone for testing. The LC-containing plasmids are constant throughout the experiments.

Alternately, overlapping PCR is used to generate inserts containing the entire IgG constant domains (Casimiro et al. (1997), Structure 5; 1407-1412). The resulting single inserts contain 15 base pair 5′ and 3′ overlaps to allow recombinase-based cloning as described above.

For each of these methods listed above, the new HC-containing vector is then transformed into Dam+E. coli (Invitrogen One Shot Top10 Chemically Competent E. coli) and plated on LB+Carbenicillin plates 37° C. overnight, Colonies were picked and mutations were verified by sequence analysis. To generate IgG BsAb protein, four plasmids, each containing either a HC or a LC from two separate MAbs, are co-transfected into 2 mL cultures of HEK293F cells using transfection reagent from Life Technologies. The plasmids are transfected at 1.3 μg of each LC and 0.67 μg of each HC into 2 mL cultures. After 5 days of shaking incubation in a CO₂ incubator at 37° C., the cell culture supernatants are collected and filtered through 0.2 μm filters. The supernatants are purified, prepared, and analyzed by high pressure liquid chromatography/mass spectrometry (LCMS) as described in Lewis et al., 2014 Nature Biotechnol. 32: 191-198. One deviation was that the proteins are enzymatically deglycosylated after purification and neutralization to approximately pH 8.0 using 1 M Tris, pH 8.5-9.0. Each protein was deglycosylated by the addition of 1 μL N-Glycanase (Prozyme) for 3-14 hrs at 37° C. prior to being submitted for LCMS.

Results

Select C_(H)3 heterodimer designs from Example 1 are constructed in the HCs listed in Table 4. The designs for testing include 7.8, 7.8.60, 20.8, 20.8.26, 20.8.34, and 20.8.37. The HCs and LCs from each antibody pair (4 chains total) are transfected into 293F, cultured for 5 days, purified using protein G capture, and analyzed by LCMS as described in the methods.

TABLE 4 Sequence ID numbers of HC and LC constructs prepared to evaluate heterodimerization potential of C_(H)3 Designs in IgG BsAb Format First Parental MAb Second Parental MAb SEQ Constructs (C_(H)3 Design SEQ ID Constructs (C_(H)3 Design ID or WT indicated)^(a,c) NO: or WT indicated)^(b,c) NO: pertuzumab HC_WT  9* BHA10 HC_WT 21 pertuzumab HC_7.8_A 10* BHA10 HC_7.8_B 22 pertuzumab HC_7.8.60_A 11* BHA10 HC_7.8.60_B 23 pertuzumab HC_20.8_A 12* BHA10 HC_20.8_B 24 pertuzumab HC_20.8.26_A 13* BHA10 HC_20.8.26_B 24 pertuzumab HC_20.8.34_A 13* BHA10 HC_20.8.34_B 25 pertuzumab HC_20.8.37_A 13* BHA10 HC_20.8.37_B 26 pertuzumab LC 14 BHA10 LC 27 MetMAb HC_WT 15* matuzumab HC_WT 28 MetMAb HC_7.8_B 16* matuzumab HC_7.8_A 29 MetMAb HC_7.8.60_B 17* matuzumab 31 HC_7.8.60_A MetMAb HC_20.8_A 18* matuzumab HC_20.8_B 33 MetMAb HC_20.8.26_A 19* matuzumab 33 HC_20.8.26_B MetMAb HC_20.8.34_A 19* matuzumab 34 HC_20.8.34_B MetMAb HC_20.8.37_A 19* matuzumab 35 HC_20.8.37_B MetMAb LC 20 matuzumab LC 36 pertuzumab HC_WT  9* matuzumab HC_WT 28 pertuzumab HC_7.8_A 10* matuzumab HC_7.8_B 30 pertuzumab HC_7.8.60_A 11* matuzumab 32 HC_7.8.60_B ^(a)The pertuzumab and MetMab HC and LC constructs also comprise the Fab Design AB2133(a) mutations as described in WO2014/150973 (HC: V_(H) _(—) Q39K, R62E; C_(H)1_H172A, F174G/LC: V_(L) _(—) D1R, Q38D; C_(L) _(—) L135Y, S176W), ^(b)The BHA10 and matuzumab HC and LC constructs also comprise the Fab Design H4WT(+DR_CS) mutations as described in WO2014/150973 (HC: V_(H) _(—) Q39Y, Q105R; C_(H)1_S127C, K228D, C230G/LC: V_(L) _(—) Q38R, K42D; C_(L) _(—) D122K), ^(c)The designation “A” following a C_(H)3 domain design number indicates that the HC contains one of the member set of C_(H)3 mutations of the given design number mutation pair, and the designation “B” indicates that the HC contains the corresponding member set of C_(H)3 mutations of the given design number. *Sequence also comprises an N297Q mutation in the CH12 domain to reduce glycosylation heterogeneity to facilitate LCMS analysis

The results of the LCMS data indicate that the exemplified C_(H)3 heterodimer designs from Example 1 which were incorporated into the IgG BsAb format resulted in improved correct IgG BsAb assembly (Table 5). Using the wild-type C_(H)3, the average percentage of heterodimer is found to be 49%—almost identical to the theoretical level expected if both HCs express equally well and there is no bias for heterodimer formation. When the designs are added to the C_(H)3 domain, similar percentages of heterodimer are observed by LCMS of the IgG BsAbs as the percentages found in Example 1 by UPLC and FRET using the MetMAb and FRET constructs.

TABLE 5 Specific Assembly of IgG BsAbs or Mis-matched Species Incorporating WT CH3 Domains or Select CH3 Designs Pertuzumab × BHA10 IgG BsAbs (with or without CH3 Designs) Pertuzumab BHA10 % AB parental Mab parental Mab (LC1/LC2) % AA % BB % AB % AB CH3 Design CH3 Design (correct IgG Homodimer Homodimer (2x LC1) (2x LC2) or WT ^(a, c) or WT ^(b, c) BsAb (incorrect (incorrect (incorrect (incorrect (HCA* + LC1) (HCB + LC) assembly) assembly) assembly) assembly) assembly) WT_A WT_B 52.6 18.5 28.9 0 0  (SEQ ID 9 + 14) (SEQ ID 21 + 27) 7.8_A 7.8_B 69.3 0 15.1 15.6 0 (SEQ ID 10 + 14) (SEQ ID 22 + 27) 7.8.60_A 7.8.60_B 95.2 0.7 1.6 0 2.5 (SEQ ID 11 + 14) (SEQ ID 23 + 27) 20.8_A 20.8_B 96.5 3.5 0 0 0 (SEQ ID 12 + 14) (SEQ ID 24 + 27) 20.8.26_A 20.8.26_B 96.9 3.1 0 0 0 (SEQ ID 13 + 14) (SEQ ID 24 + 27) 20.8.34_A 20.8.34_B 90.3 0 0 9.7 0 (SEQ ID 13 + 14) (SEQ ID 25 + 27) 20.8.37_A 20.8.37_B 89.6 2.9 0 7.5 0 (SEQ ID 13 + 14) (SEQ ID 26 + 27) MetMAb × Matuzumab IgG BsAbs (with or without CH3 Designs) MetMAb Matuzumab % AB parental MAb parental MAb (LC1/LC2) % AA % BB % AB % AB CH3 Design CH3 Design (correct IgG Homodimer Homodimer (2x LC1) (2x LC2) or WT ^(a, c) or WT ^(b, c) BsAb (incorrect (incorrect (incorrect (incorrect (HCA* + LC1) (HCB + LC) assembly) assembly) assembly) assembly) assembly) WT_A WT_B 42 0 58 0 0 (SEQ ID 15 + 20) (SEQ ID 28 + 36) 7.8_B 7.8_A* 100 0 0 0 0 (SEQ ID 16 ± 20) (SEQ ID 29 + 36) 7.8.60_B 7.8.60_A* 90.8 0 0 9.2 0 (SEQ ID 17 + 20) (SEQ ID 31 + 36) 20.8_A 20.8_B 75.6 0 0 0 24.4 (SEQ ID 18 + 20) (SEQ ID 33 + 36) 20.8.26_A 20.8.26_B 58.6 8.1 0 0 33.3 (SEQ ID 19 + 20) (SEQ ID 33 + 36) 20.8.34_A 20.8.34_B 95.8 0 0 0 4.2 (SEQ ID 19 + 20) (SEQ ID 34 + 36) 20.8.37_A 20.8.37_B 79.1 0 0 0 20.9 (SEQ ID 19 + 20) (SEQ ID 35 + 36) Pertuzumab × Matuzumab IgG BsAbs (with or without CH3 Designs) Pertuzumab Matuzumab % AB parental MAb parental MAb (LC1/LC2) % AA % BB % AB % AB CH3 Design CH3 Design (correct IgG Homodimer Homodimer (2x LC1) (2x LC2) or WT ^(a, c) or WT ^(b, c) BsAb (incorrect (incorrect (incorrect (incorrect (HCA* + LC1) (HCB + LC) assembly) assembly) assembly) assembly) assembly) WT_A WT_B 53.1 26.9 20 0 0 (SEQ ID 9 + 14) (SEQ ID 28 + 36) 7.8_A 7.8_B 95.4 0.5 2.9 0 1.2 (SEQ ID 10 + 14) (SEQ ID 30 + 36) 7.8.60_A 7.8.60_B 98.1 0.6 1.3 0 0 (SEQ ID 11 + 14) (SEQ ID 32 + 36) 20.8_A 20.8_B 96 2.1 2 0 0 (SEQ ID 12 + 14) (SEQ ID 33 + 36) 20.8.26_A 20.8.26_B 95.2 1.8 2.9 0 0 (SEQ ID 13 + 14) (SEQ ID 33 + 36) 20.8.34_A 20.8.34_B 96.9 1.5 1.6 0 0 (SEQ ID 13 + 14) (SEQ ID 34 + 36) 20.8.37_A 20.8.37_B 97.6 2.0 0.5 0 0 (SEQ ID 13 + 14) (SEQ ID 35 + 36) ^(a) The pertuzumab and MetMab HC and LC constructs also comprise the Fab Design AB2133(a) mutations as described in WO2014/150973 (HC: V_(H) _(—) Q39K, R62E; C_(H)1 _(—) H172A, F174G/LC: V_(L) _(—) D1R, Q38D; C_(L) _(—) L135Y, S176W), ^(b) The BHA10 and matuzumab HC and LC constructs also comprise the Fab Design H4WT(+DR _(—) CS) mutations as described in WO2014/150973 (HC: V_(H) _(—) Q39Y, Q105R; C_(H)1 _(—) S127C, K228D, C230G/LC: V_(L) _(—) Q38R, K42D; C_(L) _(—) D122K), ^(c) The designation “A” following a CH3 domain design number indicates that the HC contains one of the member set of CH3 mutations of the given design number mutation pair, and the designation “B” indicates that the HC contains the corresponding member set of CH3 mutations of the given design number. *Heavy chain SEQ ID NOs 9-13 and 15-19 also comprises an N297Q mutation in the CH2 domain to reduce glycosylation heterogeneity to facilitate LCMS analysis Conclusions

The data in Table 5 clearly demonstrates that designs 7.8, 7.8.60, 20.8, 20.8.26, 20.8.34 and 20.8.37 improve the assembly of the desired heterotetrameric IgG BsAbs (i.e., HCA/LC1+HCB/LC2) over what was observed with the WT C_(H)3 s. The strong correlation between the % heterodimer induced by each design described in Example and the % heterodimer induced within the IgG BsAbs in Example 2 suggests that all of the exemplified designs from Example 1 that improved heterodimer formation based on the UPLC and FRET assays will improve the percentage of heterodimer within the IgG BsAbs format.

Example 3 Complete IgG Bispecific Antibodies (IgG4 HC Backbones) Comprising C_(H)3/C_(H)3 Interface Modifications

Using the same parental Mab pairs as described in Example 2, complete IgG Bispecific Antibodies comprising select C_(H)3 designs and fully IgG4 constant domains in each heavy chain are constructed. As in Example 2, the C_(H)3 designs are incorporated into the C_(H)3 domains of each parental Mab pair, with each Mab C_(H)3 domain receiving one set of mutations of a particular design pair (i.e, either the A Chain or B Chain mutations), and the other Mab C_(H)3 domain receiving the other set of mutations of the design pair. Each HC and LC prepared also included the previously described mutations in the Fab region to promote proper HC-LC pairing as well. Matuzumab and BHA10 HCs and LCs contain Design H4WT (+DR_CS), while the pertuzumab and MetMAb HCs and LCs contain Design AB2133(a), each as described in WO2014/150973 (see also, Lewis et al. (2014), Nature Biotech. 32; 191-198 (Designs VRD2, VRD1 and CRD2). Further, to make the resulting IgG BsAb proteins more homogeneous and amenable to eventual LCMS analyses, serine 241 (Kabat Numbering) was mutated to proline (S241P) to reduce natural IgG4 half-antibody formation (see, Aalberse, R. C. and Schuurman, J. Immunology, 105, 9-19 (2002)). Additionally, asparagine 297 was mutated to glutamine (N297Q) to eliminate N-linked glycosylation. Lastly, the IgG4 lower hinge regions contain a double alanine mutation at positions 234 and 235 that have been previously described.

Molecular Biology

DNA encoding complete IgG4 constant domain regions, containing both the Fab (C_(H)1) specificity designs and C_(H)3 hetero-dimerization designs, are constructed in separate pieces or “blocks” as follows. A DNA block coding for the human IgG4 C_(H)1 region is prepared which contains a 5′ region overlapping with an NheI restriction site located behind the variable domain-encoding regions in the expression cassette. A second DNA block coding for the human IgG4 C_(H)2-C_(H)3 region, containing a BSU361 restriction site at the beginning of the C_(H)2 encoding region, a PshAI site at the 3′ end of the C_(H)2 encoding region, and a 3′ region overlapping within the EcoRI site of a template IgG1 expression cassette is also prepared. Two C_(H)2-C_(H)3 DNA blocks are prepared for each heavy chain of each parental Mab, one containing the hetero-dimerization design 7.8.60 mutations (either the “A” or “B” side mutations) and the other containing the design 20.8.34 mutations (either the “A” or “B” side mutations) The pertuzumab and metMAb constructs are designed to contain the “AB2133a” encoding Fab (C_(H)1) design mutations and the ‘A’ side mutations for either 7.8.60 or 20.8.34, whereas the matuzumab and BHA10 constructs are designed to contain the “H4WT(+DR_CS)” encoding Fab (C_(H)1) designs and the ‘B’ side mutations for either 7.8.60 or 20.8.34. Overlapping PCR is performed with the C_(H)1 and the C_(H)2-C_(H)3 DNA blocks to generate inserts containing the entire human IgG4 constant domains (Casimiro et al, (1997), Structure 5; 1407-1412). The complete IgG4 constant domain constructs are then amplified prior to cloning into mammalian expression vectors.

Mammalian expression plasmids encoding human IgG1 heavy chains for each of pertuzumab, metMab, matuzumab, and BHA10, as previously described (Lewis et. Al, Nat. Biotechnol, 32(2); 191-198 (2014)), are cut at restriction sites (NheI and EcoRI) at the 5′ and 3′ ends of the heavy chain constant domain coding region to allow excision of the IgG1 constant domain-encoding sequences. The linearized vectors are then purified using a DNA gel extraction kit (Qiagen, Cat. No. 28706) according to the manufacturer's protocol. The human IgG4 constant domain encoding constructs are then cloned into the previously cut expression plasmid using Gibson Assembly® Master Mix (New England Biolabs). All constructs utilized a murine kappa leader signal sequence that is cleaved upon secretion. Ligated constructs are transformed into chemically competent Top 10 E. Coli cells (Life Technologies) for scale up. Colonies are selected using an ampicillin selection marker, cultured, and final plasmids are prepared (Qiagen Mini Prep Kit). Correct sequences are confirmed by in-house DNA sequencing.

Complete IgG BsAbs are expressed in HEK293F cells as described in Example 2 above and as provided in Lewis et al., cited above. The heavy chain and light chain components of the complete IgG bispecific antibodies, constructed in IgG4 heavy chain backbones, and their corresponding sequences, are provide in Table 6 below.

TABLE 6 Sequence ID numbers of HC and LC constructs prepared to evaluate heterodimerization potential of C_(H)3 Designs in IgG BsAb Format (IgG4 HC backbone) First Parental MAb Second Parental MAb SEQ Constructs (C_(H)3 Design SEQ ID Constructs (C_(H)3 Design ID or WT indicated)^(a, c) NO: or WT indicated)^(b, c) NO: pertuzumab HC_7.8.60_A 47* BHA10 HC_7.8.60_B 56* pertuzumab 48* BHA10 HC_20.8.34_B 57* HC_20.8.34_A pertuzumab LC 49  BHA10 LC 58  MetMAb HC_7.8.60_A 50* matuzumab HC_7.8.60_B 53* MetMAb HC_20.8.34_A 51* matuzumab 54* HC_20.8.34_B MetMAb LC 52  matuzumab LC 55  ^(a)The pertuzumab and MetMab HC and LC constructs also comprise the Fab Design AB2133(a) mutations as described in WO2014/150973 (HC: V _(H—) 39K, 62E; C_(H)1_172A, 174G/LC: V_(L—)1R, 38D; C_(L) _(—) 135Y, 176W), ^(b)The BHA10 and matuzumab HC and LC constructs also comprise the Fab Design H4WT(+DR_CS) mutations as described in WO2014/150973 (HC: V_(H) _(—) 39Y, 105R; C_(H)1_127C, 228D, 230G/LC: V_(L) _(—) 38R, 42D; C_(L) _(—) 122K), ^(c)The designation “A” following a C_(H)3 domain design number indicates that the HC contains one of the member set of C_(H)3 mutations of the given design number mutation pair, and the designation “B” indicates that the HC contains the corresponding member set of C_(H)3 mutations of the given design number. *Sequence also comprises 297Q mutation (EU Index Numbering), a 241P mutation (Kabat Numbering) and a 234A, 235A double mutation (EU Index Numbering) UPLC Purification and Mass Spectromeric Analysis of IgG Bispecific Antibodies

After a five day culture, small scale purifications of prepared IgG BsAbs (in human IgG4 heavy chain backbones) from 450 μL mammalian cell culture supernatants are performed using a multidimensional Dionex UPLC system. A protein G column (POROS® G 20 μm Column, 2.1×30 mm, 0.1 mL part #. 2-1002-00) is equilibrated with 1×PBS prior to sample load. 450 μL of each cell culture supernatant (filtered using 0.2 μM syringe filters, Millipore) are injected onto the protein G column. After washing with 1×PBS, the BsAbs are eluted with 100 mM sodium phosphate, pH 2.2 (2 minutes at 1 ml/min). Titers are determined using the ultraviolet peak area at 280 nm upon elution, with calculations based upon a standard curve created with an in-house mAb. Protein G eluted peak samples are collected into vials in an autosampler held at ambient temperature.

Mass spectrometry is used to quantify bispecific antibody assembly from the purified samples. Experiments are performed using Q-ToF (Waters Technologies) mass spectrometer (MS) with a Xevo source. Samples are introduced into the MS using an Acquity UPLC system (Waters Technologies) connected in-line with a Reversed Phase column (ThermoScientific, Proswift™, RP-4H, 1×50 mm i.d.) at a flow rate of 200 uL/min. To eliminate salts and non-volatile buffers not compatible with MS, gradient elution was performed using 0.1% formic acid in H₂O (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B). Mass spectrometry is accomplished in positive ion mode with 2.6 kV capillary voltage at a 150° C. source temperature. Data processing and interpretation of LC-MIS runs is done in BiopharmLynx (a MassLynx Software application manager) using spectral summation over the chromatographic elution profile of the antibody.

The peak areas of the deconvoluted mass spectra are used to calculate the percent of each species, with the expectation that each of the IgG4 BsAb proteins with a mass near 145 kDa (whether assembled correctly or misassembled) are ionized with a similar efficiency. Results are provided in Table 7, below.

TABLE 7 Specific Assembly of IgG BsAbs (IgG4 HC backbone) or Mis-matched Species Incorporating Select CH3 Designs Pertuzumab × BHA10 IgG BsAbs (with select CH3 Designs) Pertuzumab BHA10 % AB parental MAb parental MAb (LC1/LC2) % AA % BB % AB % AB CH3 Design CH3 Design (correct IgG Homodimer Homodimer (2x LC1) (2x LC2) or WT^(a,c) or WT^(b,c) BsAb (incorrect (incorrect (incorrect (incorrect (HCA* + LC1) (HCB* + LC2) assembly) assembly) assembly) assembly) assembly) 7.8.60_A 7.8.60_B 95.8 ± 3.7 0.0 ± 0.0 4.2 ± 3.7 0.0 ± 0.0 0.0 ± 0.0 (SEQ ID 47 + 49) (SEQ ID 56 + 58) 20.8.34_A 20.8.34_B 94.8 ± 0.6 5.2 ± 0.6 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 (SEQ ID 48 + 49) (SEQ ID 57 + 58) MetMAb × Matuzumab IgG BsAbs (with select CH3 Designs) MetMAb Matuzumab % AB parental MAb parental MAb (LC1/LC2) % AA % BB % AB % AB CH3 Design CH3 Design (correct IgG Homodimer Homodimer (2x LC1) (2x LC2) or WT^(a,c) or WT^(b,c) BsAb (incorrect (incorrect (incorrect (incorrect (HCA* + LC1) (HCB* + LC2) assembly) assembly) assembly) assembly) assembly) 7.8.60_A 7.8.60_B 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 (SEQ ID 50 + 52) (SEQ ID 53 + 55) 20.8.34_A 20.8.34_B 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 (SEQ ID 51 + 52) (SEQ ID 54 + 55) Pertuzumab × Matuzumab IgG BsAbs (with select CH3 Designs) Pertuzumab Matuzumab % AB parental MAb parental MAb (LC1/LC2) % AA % BB % AB % AB CH3 Design CH3 Design (correct IgG Homodimer Homodimer (2x LC1) (2x LC2) or WT^(a,c) or WT^(b,c) BsAb (incorrect (incorrect (incorrect (incorrect (HCA* + LC1) (HCB* + LC2) assembly) assembly) assembly) assembly) assembly) 7.8.60_A 7.8.60_B 90.9 ± 1.7 9.1 ± 1.7 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 (SEQ ID 47 + 49) (SEQ ID 53 + 55) 20.8.34_A 20.8.34_B 94.1 ± 3.7 2.3 ± 2.2 3.6 ± 1.5 0.0 ± 0.0 0.0 ± 0.0 (SEQ ID 48 + 49) (SEQ ID 54 + 55) ^(a)The pertuzumab and MetMab HC and LC constructs also comprise the Fab Design AB2133(a) mutations as described in WO2014/150973 (HC: V_(H) _(—) 39K, 62E; C_(H)1_172A, 174G/LC: V_(L) _(—) 1R, 38D; C_(L) _(—) 135Y, 176W), ^(b)The BHA10 and matuzumab HC and LC constructs also comprise the Fab Design H4WT(+DR_CS) mutations as described in WO2014/150973 (HC: V_(H) _(—) 39Y, 105R; C_(H)1 _(—) 127C, 228D, 230G/LC: V_(L) _(—) 38R, 42D; C_(L) _(—) 122K), ^(c)The designation “A” following a CH3 domain design number indicates that the HC contains one of the member set of CH3 mutations of the given design number mutation pair, and the designation “B” indicates that the HC contains the corresponding member set of CH3 mutations of the given design number. *Heavy chain SEQ ID NOs 47, 48, 50, 51, 53, 54, 56 and 57 also comprise a 297Q mutation (EU index Numbering), a 241P mutation (Kabat Numbering) and a 234A 235A double mutation (EU index Numbering) Conclusions

The data in Table 7 demonstrates that designs 7.8.60 and 20.8.34, when applied to the human IgG4 constant domains, and paired with the Fab designs, induce predominantly correct assembly (>90%) of the desired heterotetrameric IgG BsAbs (i.e., HCA/LC1+HCB/LC2) over the misassembled protein products. No LC mispairing (existence of two of the same LCs on a HC heterodimer) was observed for any of the IgG BsAbs in the human IgG4 heavy chain backbones. Small levels of homodimeric HC products were observed (either AA homodimer or BB homodimer), however, the clear main peak for each of the six BsAbs prepared was the desired IgG BsAb.

Sequences SEQ ID. NO: 1 (5D5 heavy chain (WT CH3)) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDP SNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSWLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 2 (Human IgG1 Fc (HA-tagged, WT CH3)) YPYDVPDYASGSGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK SEQ ID. NO: 3 (5D5 light chain) DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIY WASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID. NO: 4 (Myc-EGFR D3-Human IgG1 Fc (WT CH3)) EQKLISEDDLSGSEERKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAF RGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQH GQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKI ISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKGSDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID. NO: 5. (Myc-mVEGFR1 D3-Human IgG1 Fc (WT CH3)) EQKLISEEDLSGSQTNTILDVQIRPPSPVRLLHGQTLVLNCTATTELNTRVQMSWN YPGKATKRASIRQRIDRSHSHNNVFHSVLKINNVESRDKGLYTCRVKSGSSFQSF NTSVHGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK SEQ ID. NO: 6. (WT Human IgG1 Fc with lower hinge) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO: 7. Human IgG1 Fc_(7.4_A CH3) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 8. Human IgG1 Fc_(7.4_B + 366M CH3) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSL M CLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYS V LWDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID. NO: 9. (Pertuzunmb HC_(AB2133a Fab + WT CH3 with N297Q)) EVQLVESGGGLVQPGGSLRLSCAASGFTFDYTMDWVR K APGKGLEWVADVNP NSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD YWVGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID NO. 10. (Pertuzumab HC_(AB2133a Fab + 7.8_A CH3 with N297Q)) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVADVNP NSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVL M SDGSFFL A SKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK SEQ ID. NO: 11. (Perturtumab HC_(AB2133a Fab + 7.8.60_A CH3 with N297Q)) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVADVNP NSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREE Q YSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT D NQVSLTCLKGFYPSDIAV EWESNGQPENNYKTTPPVL M SDGSFFL A SKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK SEQ ID. NO: 12. (Pertuzumab HC (AB2133a Fab +_20.8_A Ch3 with N297Q)) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVADVNP NSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTGVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQV S TLPPSREEMTKNQVSL V CLV Y GFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 13. (Pertuzumab HC_(AB2133a Fab + 20.8.26_A, 20.8.34_A or 20.8.37_A CH3 with N297Q) ) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVADVNP NSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQV S TLPPSREEMTKNQVSL M CLV Y GFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 14. (Pertuzumab LC (AB2133a Fab)) R IQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRY TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGQPK AAPSVTLFPPSSEELQANKATLVC Y ISDFYPGAVTVAWKADSSPVKAGVETTTPS KQSNNKYAA W SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC SEQ ID. NO: 15. (Met HC_(AB2133a Fab + WT CH3 with N297Q)) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGMIDP SNSDTRFNP E FKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 16. (Met HC_(AB2133a Fab + 7.8_B CH3 with N297Q)) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGMIDP SNSDTRFNP E FKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL V CLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 17. (Met HC_(AB2133a Fab + 7.8.60_B CH3 with N297Q)) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGMIDP SNSDTRFNP E FKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPR R P R VYTLPPSREEMTKNQVSL V CLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 18. (Met HC_(AB2133a Fab + 20.8_A CH3 with N297Q)) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGMIDP SNSDTRFNP E FKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQV S TLPPSREEMTKNQVSL V CLV Y GFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 19. (Met HC (AB2133a Fab + 20.8.26_A, 20.8.34_A or 20.8.37_A CH3 with N297Q)) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGMIDP SNSDTRFNP E FKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNIKVDKICVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQV S TLPPSREEMTKNQVSL M CLV Y GFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 20. (Met LC (AB2133 Fab)) R IQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQ D KPGKAPKLLIY WASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKV EIKGQPKAAPSVTLFPPSSEELQANKATLVC Y ISDFYPGAVTVAWKADSSPVKAG VETTTPSKQSNNKYAA W SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC SEQ ID. NO: 21. (BHA10 HC (H4WT + DR_CS Fab + WT CH3)) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVR Y APGQGLEWMGWIY PGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYW G R GTTVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID. NO:22. (BHA10 HC (H4WT +DR_CS Fab + 7.8_B CH3)) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVR Y APGQGLEWMGWIY PGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYW G R GTTVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKTKDTLMISRTPEVTGVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL V CLVKGFYPSDIAVE WESNGQPENNYKTTTPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 23. (BHA10HC (H4WT + DR_CS Fab + 7.8.60_B CH3)) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVR Y APGQGLEWMGWIY PGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYW G R GTTVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPR R P R VYTLPPSREENYTKNQVSL V CLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 24. (BHA10 HC (H4WT + DRCS Fab + 20.8_B or 20.8.26_B CH3)) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVR Y APGQGLEWMGWIY PGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYW G R GTTVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWENGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRE D MTKNQV Q LTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 25. (BHA10 HC (H4WT + DR_CS Fab + 20.8.34_B CH3)) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVR Y APGQGLEWMGWIY PGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYW G R GTTVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSR GD MTKNQV Q LTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 26. (BHA10 HC (H4WT + DR_CS Fab + 20.8.37_B CH3)) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVR Y APGQGLEWMGWIY PGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYW G R GTTVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRE D MTKNQV R LTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID. NO: 27. (BHA10 LC (H4WT + DR_CS Fab)) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQ R KPG D APKSLISSASYRY SGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKRTVA APSVFIFPPS K EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVEEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID. NO: 28. (Matuzumab HC (H4WT + DR_CS Fab + WT CH3)) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFN PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG RYFDYWG R GTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVWSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK SEQ ID. NO: 29. (Matuzumab HC (H4WT + DR_CS Fab + 7.8_A CH3)) QVQLVQSGAEVKKKGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFN PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG RYFDYWG R GTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVL M SDGSFFL A SKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK SEQ ID. NO: 30. (Matuzumab HC (H4WT + DR_CS Fab + 7.8_B CH3)) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFN PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG RYFDYWG R GTLVTVSSASITCGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL V CLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK SEQ ID. NO: 31. (Matuzumab HC (H4WT + DR_CS Fab + 7.8.60_A CH3)) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFN PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG RYFDYWG R GTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICKVNHKPSNTKV DKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT D NQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVL M SDGSFFL A SKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK SEQ ID. NO: 32. (Matuzumab HC (H4WT + DR_CS Fab + 7.8.60_B CH3)) QVQLVQSGAEVKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFN PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRRSEDTAVYYCASRDYDYDG RYFDYWG R GTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSEGTQTYICNVNHKPSNTKV DKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPR R P R VYTLPPSREEMTKNQVSL V CLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK SEQ ID. NO: 33. (Matuzumab HC (H4WT + DR_CS Fab + 20.8_B or 20.8.26_B CH3)) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFN PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG RYFDYWG R GTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE D MTKNQV Q LTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK SEQ ID. NO: 34. (Matuzumab HC (H4WT + DR_CS Fab + 20.8.34_B CH3)) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFN PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG RYFDYWG R GTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVFP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREHQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR G D MTKNQV Q LTCLVKGFYP SDIAVEWESNGQPENNTKTTPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK SEQ ID. NO: 35. (Matuzumab HC (H4WT + DR_CS Fab + 20.8.37_B CH3)) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFN PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG RYFDYWG R GTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVFP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREHQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE D MTKNQV R LTCLVKGFYP SDIAVEWESNGQPENNTKTTPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK SEQ ID. NO: 36. (Matuzumab LC (H4WT + DR_CS Fab)) DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQ R KPG D APKLLIYDTSNLAS GVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVAAP SVFIFPPS K EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Fc Domain (CH2-CH3) Sequences SEQ ID NO: 37. Human IgG1 Fc_(WT) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSTFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 38. Human IgG1 Fc_(7.8_A CH3) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVL M SDGSFFL A SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 39. Human IgG1 Fc_(7.8.60_A CH3) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYNTDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMT D NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVL M SDGSFFL A SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 40. Human IgG1 Fc_(20.8_A, 20.8.31_A or 20.8.33_A CH3) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQV S TLPPSREEMTKNQVSL V CLV Y GFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSISPGK SEQ ID NO: 41. Human IgG1 Fc_(20.8.26_A, 20.8.34_A or 20.8.37_A CH3) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQV S TLPPSREEMTKNQVSL M CLV Y GFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 42. Human IgG1 Fc_(7.8_B or 7.4_B CH3) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLTSREEMTKNQVSL V CLCKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYS V LTVDKSRWQQGNYFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 43. Human IgG1 Fc_(7.8.60_B CH3) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVE NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQP R R P RVYTLPPSREEMTKNQVSL V CLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYS V LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 44. Human IgG1 Fc_(20.8_B or 20.8.26_B CH3) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRE D MTKNQV Q LTCLVKGFYRSDIAVEWESNGQPENNYKT TPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 45. Human IgG1 Fc_(20.8.33_B or 20.8.34_B CH3) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPSR GD MTKNQV Q LTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 46. Human IgG1 Fc_(20.8.31_B or 20.8.37_B CH3) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRE D MTKNQV R LTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFL A SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 47. (Pertuzumab IgG4 HC (AB2133a Fab + 7.8.60_A CH3 with 241P (Kabat Numbering), 234A, 235A, 297Q mutations (EU Index Numbering)) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVADVNP NSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDKKPSNTKVDKR VESKYGPPCP P CPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQF Q STYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT D NQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVL M SDGSFFL A SRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLG SEQ ID NO: 48. (Pertuzumab IgG4 HC (AB2133a Fab + 20.8.34_A CH3 with 241P (Kabat Numbering), 234A, 235A, 297Q mutations (EU Index Numbering)) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVADVNP NSGGSIYNQ E FKGRFTLSYDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFD YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR VESKYGPPCP P CPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTFCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQF Q STYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQV S TLPPSQEEMTKNQVSL M CLV Y GFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLG SEQ ID NO: 49. (Pertuzumab LC (2133a Fab)) R IQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRY TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGQPK AAPSVTLFPPSSEELQANKATLVC Y ISDFYPGAVTVAWKADSSPVKAGVETTTPS KQSNNKYAA W SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC SEQ ID NO: 50. (MetMAb IgG4 HC (AB2133a Fab + 7.8.60_A CH3 with 241P (Kabat Numbering), 234A, 235A, 297Q mutations (EU Index Numbering)) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGMIDP SNSDTRFNP E FKDRFTISADTSKNTAYLQMNSIRAEDTAVYYCATYRSYVTPLDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQF Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQVYTLPPSQEEMT D NQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVL M SDGSFFL A SRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLG SEQ ID NO: 51. (MetMAb IgG4 HC (AB2133a Fab + 20.8.34_A CH3 with 241P (Kabat Numbering), 234A, 235A, 297Q mutations (EU Index Numbering)) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVGMIDP SNSDTRFNP E FKDRFTISADTSKNTAYLQMNSIRAEDTAVYYCATYRSYVTPLDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCP P CPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQF Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQV S TLPPSQEEMTKNQVSL M CLV Y GFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLG SEQ ID NO: 52. (MetMAb LC (AB2133a Fab)) RIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQDKPGKAPKLLIY WASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKV EIKGQPKAAPSVTLFPPSSEELQANKATLVC Y ISDFYPGAVTVAWKADSSPVKAG VETTTPSKQSNNKYAA W SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC SEQ ID NO: 53. (Matuzumab IgG4 HC (H4WT (Fab + 7.8.60_B CH3 with 241P (Kabat Numbering), 234A, 235A, 297Q mutations (EU Index Numbering)) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFN PSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDG RYFDYWG R GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKV DKRVES D YGPPCP P CPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQF Q STYRVVSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPR R P R VYTLPPSQEEMTKNQVSL V CLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG SEQ ID NO: 54. (Matuzumab IgG4 HC (H4WT(Fab + 20.8.34_B CH3 with 241P (Kabat Numbering), 234A, 235A, 297Q mutations (EU Index Numbering)) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFN PSNGRTNYNEKFKSKATMTVDTSTWAYMELSSLRSEDTAVYYCASRDYDYDG RYFDYWG R GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKV DKRVES D YGPPCP P CPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQE Q STYRVVSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ GD MTKNQV Q LTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFL A SRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG SEQ ID NO: 55. (Matuzumab LC (H4WT(+DR_CS)Fab)) DIQMTQSPSSLSASVGDRVTITCSASSSVTYMWYQ R KPG D APKLLIYDTSNLAS GVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTGQGTKVEIKRTVAAP SVFIFPPS K EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 56. (BHA10 IgG4 HC (H4WT(Fab + 7.8.60_B CH3 with 241P (Kabat Numbering), 234A, 235A, 297Q mutations (EU Index Numbering)) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVR Y APGQGLEWMGWIY PGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYW G R GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES D YGPPCP P CPAPE AA GGPSVELFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQF Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPR R P R VYTLPPSQEEMTKNQVSL V CLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYS V LTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLG SEQ ID NO: 57. (BHA10 IgG4 HC (H4WT(+DR_CS)Fab + 20.8.34_B CH3 with 241P (Kabat Numbering), 234A, 235A, 297Q mutations (EU Index Numbering)) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTYYLHWVRYAPGQGLEWMGWIY PGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYW GRGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFREPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES D YGPPCP P CPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQF Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQ GD MTKNQV Q LTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLASRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLG SEQ ID NO: 58. (BHA10 LC (H4WT(+DR_CS)Fab)) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQ R KPG D APKSLISSASYRY SGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKRTVA APSVFIFPPS K EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC IgG4 Fc Domain (CH2-CH3) Sequences SEQ ID NO: 59. Human IgG4 Fc_(WT) APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGEPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 60. Human IgG4 Fc_(7.4_A CH3) APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFL A SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 61. Human IgG4 Fc_(7.8_A CH3) APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQPWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVL M SDGSFFL A SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 62. Human IgG4 Fc_(7.8.60_A CH3) APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQENSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMT D NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVL M SDGSFFL A SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSEGK SEQ ID NO: 63. Human IgG4 Fc_(20.8_A, 20.8.31_A or 20.8.33_A CH3) APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQV S TLPPSQEEMTKNQVSL V CLV Y GFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYS V LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSWK SEQ ID NO: 64. Human IgG4 Fc_(20.8.26_A, 20.8.34_A or 20.8.37_A CH3) APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVSTLPPSQEEMTKNQVSL M CLV Y GFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYS V LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 65. Human IgG4 Fc_(7.8_B or 7.4_B CH3) APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSL V CLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYS V LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 66. Human IgG4 Fc_(7.8.60_B CH3) APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPR R P R VYTLPPSQEEMTKNQVSL V CLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYS V LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSWK SEQ ID NO: 67. Human IgG4 Fc_(20.8_B or 20.8.26_B CH3) APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQE D MTKNQV Q LTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFL A SRLTVDKSRWQEGNVSCSVMHEALHNHYTQKSLSLSWK SEQ ID NO: 68. Human IgG4 Fc_(20.8.33_B or 20.8.34_B CH3) APEFLGGPSVFLFPPKYKDTLIMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQ GD MTKNQV Q LTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFL A SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 69. Human IgG4 Fc_(20.8.3_B or 20.8.37_B CH3) APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQE D MTKNQV R LTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFL A SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLKSLGK (Bold underlined residues represent mutations to parental Mab or WT sequence) 

We claim:
 1. A bispecific antibody comprising: a. a first heavy chain, wherein said first heavy chain comprises a first heavy chain variable domain (V_(H)) and a first human IgG heavy chain constant region, wherein said first human IgG heavy chain constant region is a human IgG1 or human IgG4 heavy chain constant region that comprises an alanine at residue 407, a methionine at residue 399, and an aspartic acid at residue 360 of the C_(H)3 domain; b. a first light chain, wherein said first light chain comprises a first light chain variable domain (V_(L)) and a first light chain constant domain (C_(L)); c. a second heavy chain, wherein said second heavy chain comprises a second V_(H) and a second human IgG heavy chain constant region, wherein said second human IgG heavy chain constant region is a human IgG1 or human IgG4 heavy chain constant region that comprises an arginine at residues 345 and 347, a valine at residue 366 and a valine at residue 409 of the C_(H)3 domain; and d. a second light chain, wherein said second light chain comprises a second V_(L) and a second C_(L).
 2. The bispecific antibody according to claim 1, wherein a. one of said first or second heavy chains further comprises a V_(H) comprising a lysine at residue 39 and a glutamic acid at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat numbering system, and a human IgG1 or human IgG4 C_(H)1 domain comprising an alanine at residue 172 and a glycine at residue 174; b. one of said first or second light chains comprises a V_(L) comprising an arginine at residue 1 and an aspartic acid at residue 38, and a C_(L) comprising a tyrosine at residue 135 and a tryptophan at residue 176; c. the other of said first or second heavy chains further comprises a V_(H) comprising a tyrosine at residue 39 and a WT human IgG1 or human IgG4 C_(H)1 domain; and d. the other of said first or second light chains comprises a V_(L) comprising an arginine at residue 38 and a WT C_(L), wherein the V_(H) domain comprising a lysine at residue 39 and a glutamic acid at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat and the human IgG1 or human IgG4 C_(H)1 domain comprising an alanine at residue 172 and a glycine at residue 174 together with the V_(L) comprising an arginine at residue 1 and an aspartic acid at residue 38 and the C_(L) comprising a tyrosine at residue 135 and a tryptophan at residue 176 form a first Fab which directs binding to a first target; and the V_(H) domain comprising a tyrosine at residue 39 and the WT human IgG1 or human IgG4 C_(H)1 domain together with the V_(L) comprising an arginine at residue 38 and the WT C_(L) domain form a second Fab which directs binding to a second target which is different from the first target.
 3. The bispecific antibody of claim 1, wherein said first human IgG heavy chain constant region comprises SEQ ID NO: 39, and said second human IgG heavy chain constant region comprises SEQ ID NO:
 43. 4. The bispecific antibody of claim 1, wherein said first human IgG heavy chain constant region comprises SEQ ID NO: 62, and said second human IgG heavy chain constant region comprises SEQ ID NO:
 66. 5. The bispecific antibody according to claim 1, wherein a. one of said first or second heavy chains further comprises a VH comprising a lysine at residue 39 and a glutamic acid at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and a human IgG1 or human IgG4 CH1 domain comprising an alanine at residue 172 and a glycine at residue 174; b. one of said first or second light chains comprises a VL comprising an arginine at residue 1 and an aspartic acid at residue 38, and a constant domain (CL) comprising a tyrosine at residue 135 and a tryptophan at residue 176; c. the other of said first or second heavy chains further comprises a VH comprising a tyrosine at residue 39 and an arginine at residue 105 and a human IgG1 or human IgG4 CH1 domain comprising a cysteine at residue 127, an aspartic acid at residue 228, and a glycine at residue 230; and d. the other of said first or second light chains comprises a variable domain (VL) comprising an arginine at residue 38 and an aspartic acid at residue 42, and a constant domain (CL) comprising a lysine at residue 122, wherein the VH domain comprising a lysine at residue 39 and a glutamic acid at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat and the human IgG1 or human IgG4 CH1 domain comprising an alanine at residue 172 and a glycine at residue 174 together with the VL comprising an arginine at residue 1 and an aspartic acid at residue 38 and the CL domain comprising a tyrosine at residue 135 and a tryptophan at residue 176 form a first Fab which directs binding to a first target; and the VH domain comprising a tyrosine at residue 39 and an arginine at residue 105 and the human IgG1 or human IgG4 CH1 domain comprising a cysteine at residue 127, an aspartic acid at residue 228, and a glycine at residue 230 together with the VL comprising an arginine at residue 38 and an aspartic acid at residue 42 and the CL domain comprising a lysine at residue 122 form a second Fab which directs binding to a second target which is different from the first target.
 6. A bispecific antibody comprising: a. a first heavy chain, wherein said first heavy chain comprises a first VH and a first human IgG heavy chain constant region, wherein said first human IgG heavy chain constant region is a human IgG1 or human IgG4 heavy chain constant region that comprises an alanine at residue 407, a glycine at residue 356, an aspartic acid at residue 357 and a glutamine at residue 364 of the C_(H)3 domain, b. a first light chain, wherein said first light chain comprises a first VL and a first CL, c. a second heavy chain, wherein said second heavy chain comprises a second VH and a second human IgG heavy chain constant region, wherein said second human IgG heavy chain constant region is a human IgG1 or human IgG4 heavy chain constant region that comprises a valine at residue 409, a methionine at residue 366, a serine at residue 349 and a tyrosine at residue 370 of the C_(H)3 domain, and d. a second light chain, wherein said second light chain comprises a second VL and a second CL.
 7. The bispecific antibody of claim 6, wherein said first human IgG heavy chain constant region comprises SEQ ID NO: 41, and said second human IgG heavy chain constant region comprises SEQ ID NO:
 45. 8. The bispecific antibody of claim 6, wherein said first human IgG heavy chain constant region comprises SEQ ID NO: 64, and said second human IgG heavy chain constant region comprises SEQ ID NO:
 68. 9. The bispecific antibody according to claim 6, wherein a. one of said first or second heavy chains further comprises a VH comprising a lysine at residue 39 and a glutamic acid at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat numbering system, and a human IgG1 or human IgG4 CH1 domain comprising an alanine at residue 172 and a glycine at residue 174; b. one of said first or second light chains comprises a VL comprising an arginine at residue 1 and an aspartic acid at residue 38, and a CL comprising a tyrosine at residue 135 and a tryptophan at residue 176; c. the other of said first or second heavy chains further comprises a VH comprising a tyrosine at residue 39 and a WT human IgG1 or human IgG4 CH1 domain; and d. the other of said first or second light chains comprises a VL comprising an arginine at residue 38 and a WT CL, wherein the VH domain comprising a lysine at residue 39 and a glutamic acid at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat and the human IgG1 or human IgG4 CH1 domain comprising an alanine at residue 172 and a glycine at residue 174 together with the VL comprising an arginine at residue 1 and an aspartic acid at residue 38 and the CL comprising a tyrosine at residue 135 and a tryptophan at residue 176 form a first Fab which directs binding to a first target; and the VH domain comprising a tyrosine at residue 39 and the WT human IgG1 or human IgG4 CH1 domain together with the VL comprising an arginine at residue 38 and the WT CL domain form a second Fab which directs binding to a second target which is different from the first target.
 10. The bispecific antibody according to claim 6, wherein a. one of said first or second heavy chains further comprises a VH comprising a lysine at residue 39 and a glutamic acid at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and a human IgG1 or human IgG4 CH1 domain comprising an alanine at residue 172 and a glycine at residue 174; b. one of said first or second light chains comprises a VL comprising an arginine at residue 1 and an aspartic acid at residue 38, and a constant domain (CL) comprising a tyrosine at residue 135 and a tryptophan at residue 176; c. the other of said first or second heavy chains further comprises a VH comprising a tyrosine at residue 39 and an arginine at residue 105 and a human IgG1 or human IgG4 CH1 domain comprising a cysteine at residue 127, an aspartic acid at residue 228, and a glycine at residue 230; and d. the other of said first or second light chains comprises a variable domain (VL) comprising an arginine at residue 38 and an aspartic acid at residue 42, and a constant domain (CL) comprising a lysine at residue 122, wherein the VH domain comprising a lysine at residue 39 and a glutamic acid at the residue which is four amino acids upstream of the first residue of HFR3 according to Kabat and the human IgG1 or human IgG4 CH1 domain comprising an alanine at residue 172 and a glycine at residue 174 together with the VL comprising an arginine at residue 1 and an aspartic acid at residue 38 and the CL domain comprising a tyrosine at residue 135 and a tryptophan at residue 176 form a first Fab which directs binding to a first target; and the VH domain comprising a tyrosine at residue 39 and an arginine at residue 105 and the human IgG1 or human IgG4 CH1 domain comprising a cysteine at residue 127, an aspartic acid at residue 228, and a glycine at residue 230 together with the VL comprising an arginine at residue 38 and an aspartic acid at residue 42 and the CL domain comprising a lysine at residue 122 form a second Fab which directs binding to a second target which is different from the first target. 